Acroloxus coloradensis is a rare and potentially glacial relict freshwater limpet species endemic to North America. However, despite its remarkable patchy distribution across the continent, only very few genetic data is available for the different populations. Here, we sequenced two standard barcoding markers for individuals from five populations to shed some light on the genetic population structure of this species. Overall, our genetic analyses revealed low genetic variation and identified three and two haplotypes for COI and 16S rRNA, respectively. The three Rocky Mountain populations differed only slightly for COI and shared the same haplotype for 16S rRNA. For both markers, no genetic variation was detected within the two populations sampled in Québec. The present genetic data suggest that A. coloradensis is indeed a relict species and that the distribution of this species was strongly affected by the last glacial retreat in North America.