Abstract The speckled dace ( Rhinichthys osculus ) is small cyprinid fish that is widespread in the Western USA. Currently treated as a single species, speckled dace consists of multiple evolutionary lineages that can be recognized as species and subspecies throughout its range. Recognition of taxonomic distinctiveness of speckled dace populations is important for developing conservation strategies. In this study, we collected samples of speckled dace from 38 locations in the American West, with a focus on California. We used RAD sequencing to extract thousands of SNPs across the genome from samples to identify genetic differences among seven California populations informally recognized as speckled dace subspecies: Amargosa, Owens, Long Valley, Lahontan, Klamath, Sacramento, and Santa Ana speckled dace. We performed principal component analysis, admixture analysis, estimated pairwise Fst, and constructed a phylogeny to explore taxonomic relationships among these groups and test if these subspecies warrant formal recognition. Our analyses show that the seven subspecies fit into three major lineages equivalent to species: western (Sacramento-Klamath), Santa Ana, and Lahontan speckled dace. Death Valley speckled dace were determined to be two lineages (Amargosa and Long Valley) within Lahontan speckled dace. Western and Lahontan speckled dace lineages had branches that can be designated as subspecies. These designations fit well with the geologic history of the region which has promoted long isolation of populations. This study highlights the importance of genetic analysis for conservation and management of freshwater fishes.