Aulopyge huegelli Heckel, 1943 (Cypriniformes, Actinopterygii) or dalmation barbelgudgeon is endemic to the Adriatic Basin. It inhabits karst rivers and lakes in Croatia and Bosnia and Herzegovina. Due to its small and fragmented distribution area it is considered endangered locally and globally. Research on this species is limited, and molecular data are scarce. The aim of this study was to help describe the structure of dalmation barbelgudgeon populations in Croatia and Bosnia and Herzegovina by analysing genes for cytochrome b and mitochondrial control region. Samples were collected at sites along the entire distribution area of this species. Total genomic DNA was isolated in the laboratory, the cytochrome b gene and part of the mitochondrial control region were amplified by polymerase chain reaction. The success of the reactions was checked by agarose gel electrophoresis, and sequencing was performed on successfully isolated samples. Phylogenetic reconstructions of populations were obtained by the methods of maximum parsimony, maximum likelihood, and median joining. Bayesian inference method was used to estimate gene flow between populations and the effective size of populations. The results show the presence of 13 cytochrome b haplotypes and 11 control region haplotypes at research sites. The species shows moderate genetic diversity and weak genetic structuring. Gene flow between populations has not been detected. Effective population sizes are small. The populations of Roški slap on the Krka river, Ruda on the Cetina river and Šujica river with the watercourses around Tomislavgrad in Duvanjsko and Livanjsko fields have been identified as evolutionarily significant units.