Ten microsatellite loci were characterized for 34 locations from roundtail chub (Gila robusta complex) to better resolve patterns of genetic variation among local populations in the lower Colorado River basin. This group has had a complex taxonomic history and previous molecular analyses failed to identify species diagnostic molecular markers. Our results supported previous molecular studies based on allozymes and DNA sequences, which found that most genetic variance was explained by differences among local populations. Samples from most localities were so divergent species-level diagnostic markers were not found. Some geographic samples were discordant with current taxonomy due to admixture or misidentification; therefore, additional morphological studies are necessary. Differences in spatial genetic structure were consistent with differences in connectivity of stream habitats, with the typically mainstem species, G. robusta, exhibiting greater genetic connectedness within the Gila River drainage. No species exhibited strong isolation by distance over the entire stream network, but the two species typically found in headwaters, G. nigra and G. intermedia, exhibited greater than expected genetic similarity between geographically proximate populations, and usually clustered with individuals from the same geographic location and/or sub-basin. These results highlight the significance of microevolutionary processes and importance of maintaining local populations to maximize evolutionary potential for this complex. Augmentation stocking as a conservation management strategy should only occur under extreme circumstances, and potential source populations should be geographically proximate stocks of the same species, especially for the headwater forms.