Rhinichthys osculus, the Speckled Dace, is one of the most ubiquitous fish in western North America. Within the Southern California region, the local taxon is known as the Santa Ana Speckled Dace. The purpose of this study was to characterize and identify polymorphic microsatellite markers for R. osculus in which twenty-three were identified through Illumina pair-end sequencing. Seven of these loci were then used to examine the patterns of genetic variation and population structure that occurred within and among the watersheds in the Southern California. The study also examined the regional relationships among Southern California, Central California and Owen’s River Valley. Analysis of the microsatellite data revealed highly significant moderate levels of population structure exist within the Southern California region (RST=0.160, p=0.001). This structure is best explained by watershed as well as isolation by distance (R2=.2286, p=0.010). Highly significant geographic structure also exists among the geographic regions of Southern California, Central Coast, and Owen’s River Valley regions (RST= 0.600, p-value=0.001) that are congruent with the regional differentiation elucidated by mtDNA sequence data. In both cases, the degree of population differentiation was correlated with isolation by distance. Utilizing this information we were able gain a better understanding of the evolutionary relationships among the Southern California populations of Santa Ana Speckled Dace. Within the Santa Ana Speckled Dace populations we examined four models to explain the geographic structure: watershed, mountain range, tributary, and isolation by distance. While all were significant, the tributary model exhibited the higher level of population structure (RST= 0.160, p-value=0.001) and a significant correlation was exhibited between geographic distance and population structure, suggesting isolation by distance may be playing a role. The results of the microsatellite analysis are congruent with an earlier broad scale analysis of mtDNA sequence data that suggests the Central California and the Owens Valley populations diverged from each other prior to the divergence of the Santa Ana Speckled Dace populations from the Colorado Basin populations, and that the Central Coast populations were not established as a result of a migration event from the Southern California populations, as was previously hypothesized. Primarily due to human activity, Santa Ana Speckled Dace habitat has become highly fragmented resulting in some populations becoming extirpated. We hope this study will guide the strategies for the conservation of the remaining populations of Santa Ana Speckled Dace and watershed management in Southern California.