The relict dace (Relictus solitarius) is an endemic minnow restricted to isolated spring systems within Butte, Goshute, Ruby and Steptoe valleys in the eastern Nevada portion of the Great Basin. It is the only native fish in these basins, but speckled dace (Rhinichthys osculus) have been introduced into some localities in Ruby Valley and, based on morphology, the two species are believed to have hybridized in those areas. We used DNA sequence data from one mitochondrial gene (cytochrome b) and one nuclear intron (S7) to determine if putative relict dace populations in Ruby Valley share mtDNA haplotypes with speckled dace, or show signs of hybridization. Results of our analyses show that 11 of 16 Ruby Valley populations sampled contain only speckled dace, and one speckled dace population exhibited signs of limited introgressive hybridization. The remaining five Ruby Valley populations that were sampled contain relict dace that remain genetically distinct from speckled dace. The integrity of the Ruby Valley relict dace populations should be actively protected, particularly in the area surrounding Ruby Lake where speckled dace have been widely introduced.