Speciation is a fundamental process responsible for generating the great diversity of life on Earth (Mayr 1963). Speciation occurs as inherent, genetically-based barriers to gene flow that evolves between formerly inter-breeding populations (Coyne and Orr 2004). The end product of speciation is the generation of new species, reproductive communities that share a common gene pool. Thus, to understand speciation we must understand how genetic differences evolve that cause populations to become reproductively isolated from one another and form different reproductive communities.The goal of my dissertation was to discern how geographic isolation interacts with host plant-related ecological adaptation to generate new species of fruit flies in the Rhagoletis cingulata sibling species group. The R. cingulata sibling species group is comprised of taxa that may have diverged through both sympatric host shifting and geographic isolation. My thesis therefore focused on R. cingulata flies as a model system for comparing how population divergence is affected by different process associated with ecological adaptation and geography. In particular, patterns of genetic differentiation were compared within and among pairs of R. cingulata populations that have likely alternately diverged via sympatric host shifting versus allopatric speciation modes. Such an analysis can provide insight into the relative rate and degree of genetic differentiation these different modes of speciation may have for generating new biodiversity, and, by inference, the speed by which they facilitate the evolution of reproductive isolation, different reproductive communities, and speciation. * In Chapter 2, I conducted conduct a microsatellite genetic survey and mtDNA analysis of the species R. cingulata (Loew) and R. indifferens (Curran) to determine whether progressive stages of geographic speciation may be evident across the collective range of these two flies. * In Chapter 3, I performed a microsatellite survey of the species R. indifferens in the Northwestern U.S. attacking its native host bitter cherry and derived host introduced domesticated sweet cherry (P. avium) to test for genetic evidence of recent sympatric host race formation. * In Chapter 4, I reported the results of microsatellite and mtDNA surveys of different species of R. cingulata group flies attacking native hosts outside the genus Prunus to determine if shifts to plants more phylogenetically distant than cherries may exert stronger ecological selection pressures, causing increased levels of genetic differentiation and population divergence. * Finally, Chapter 5 constituted part of my studies in graduate school directed at GLOBES interdisciplinary research in which I employ an applied genetics approach to determine the identity of an unidentified infestation of mystery Rhagoletis larvae found to be infesting apple fruit in a pest quarantine area of Washington (WA) state. The results for microsatellites in Chapter 2 indicated that the eastern cherry fruit fly Rhagoletis cingulata grades into the western cherry fruit fly Rhagoletis indifferens in an inverted arch running through northern Mexico and the southwestern U.S. The pattern for microsatellites was consistent with the view of cherry flies having recently formed an interconnected series of populations (a ring-like spatial distribution) across North America than representing a single ancestral populations having been split into two large allopatric demes. However, geographic variation for mtDNA differed from that displayed by the microsatellites. The mtDNA implied a different history in which southwestern U.S. and Mexican populations of R. cingulata were diverged from cherry flies elsewhere, while R. cingulata in the East and R. indifferens in the West were virtually identical. Three explanations could explain the difference for mtDNA, including differential lineage sorting, a past north-versus-south break in the population structure of cherry flies, and the occurrence of cytoplasmic effects, possibly associated with the endosymbiotic bacteria Wolbachia. Evidence is presented that supports the possibility of cytoplasmic incompatibility in cherry flies. Thus, both geography and cytoplasmic effects may be contributing to population divergence in cherry fruit flies, however, the rate at which cytoplasmic effects are generating reproductive isolation may exceed that of standard population divergence accumulating in allopatry. The results from Chapter 3 were negative. Little evidence for host-related microsatellite divergence was observed. In addition, there was no strong evidence for the microsatellites to be associated with adult eclosion time, a trait that has been found to correlated with host-related divergence in other Rhagoletis flies. In Chapter four, pronounced microsatellite, as well as mtDNA, divergence was found among host-associated populations of R. cingulata, R. indifferens, R. turpiniae, R. chionanthi and R. osmanthi. Thus, it would appear that host ecology can exert strong selective pressures on R. cingulata group flies and help initiate speciation. However, divergent ecology primarily involves shifts between cherry and non-cherry host species, and not cherry to cherry shifts. The extent of host-related divergence observed for R. turpiniae, R. chionanthi and R. osmanthi was as great as or greater than that observed across the entire geographic range of cherry-infesting flies in North America, as well as that associated with possible cytoplasmic incompatibility in Mexico. It would therefore appear that host ecology can act on a comparatively rapid timescale to generate divergence in the group, at least for the olive infesting flies R. chionanthi and R. osmanthi. Finally, in the GLOBES interdisciplinary Chapter Five, information generated from the North American microsatellite survey of cherry fly populations was used to address an applied question to determine the identity of unknown Rhagoletis larvae that were discovered in the annual field survey of the United States Department of Agriculture and the Washington State Department of Agriculture to be infesting feral Chinese crabapple Malus spectabilis (Aiton) Borkhausen, near a commercial apple orchard in Kennewick, Benton County, Washington in 2011. The unknown fly larvae were determined to be R. indifferens and not R. pomonella, averting an unneeded and costly quarantine. The work allowed me to explore how I can apply genetic analysis to address important question of economic and social concern with partners in the general community, expanding the significance of my research beyond academic issues concerning evolutionary biology. In the concluding Chapter 6 of the thesis, a summary of the results of the research is presented and their significance is discussed. Avenues for future work are also explored that can build upon the current findings to help resolve that mystery of mystery concerning the origins of new species.