Rhinichthys osculus (Cyprinidae), the speckled dace, is the most widely distributed freshwater fish in the western United States. The southern California populations of R. osculus are identified as the Santa Ana speckled dace (SASD), though the SASD has not yet been formally recognized as a distinct taxon. Current mtDNA analysis performed in the Metcalf Lab has shown a reciprocally monophyletic relationship among three California regions; southern, central coast, and Owens Valley. Similarly, microsatellite genotyping has shown significant levels of geographic population structure. The purpose of this study was to provide nuclear DNA sequence data to determine the taxonomic status of the SASD to elucidate their evolutionary history and the relationships among the three regions, and to further define their evolutionary trajectory by comparing SASD sequence data to that of speckled dace from the Colorado River of Arizona. To examine this, three EPIC intron markers were sequenced on 54 samples representing all four regions. Based on the mtDNA and microsatellite data alone, there is strong support that the southern California populations of R. osculus are a reproductively isolated taxon at the species level. My study confirms this by showing the SASD to be reciprocally monophyletic for nuclear DNA markers, in conjunction with the mitochondrial DNA marker analyses. Because they are evolutionarily independent and face increased incidence of drought, fire, and flood, endangered species status should be considered.