Leptobarbus hoevenii is an economically significant fish found in Malossia and worldwide that has been introduced to China owing to its high economic value and good aquaculture prospects. However, the existing culture population has been unselected and degraded in germplasm, generating a need for genetic breeding- related studies on L. hoevenii. Since karyotyping and phylogenetic analysis play an important role in the conservation and utilization of fish germplasm resources, the karyotype of L. hoevenii was prepared by the chromosome cold-drop method. In order to study the evolutionary and interspecific genetic relationship among 14 species of the Barbinae subfamily, the mitochondrial 16S rRNA gene sequences were compared and used to construct phylogenetic trees. The karyotype results of L. hoevenii showed a diploid chromosome number of 2n=50, containing 8 metacentric (m) chromosomes, 14 submetacentric (sm) chromosomes, 2 subtelocentric (st) chromosomes, and 1 telocentric (t) chromosome. Furthermore, the number of chromosome arms (NF) was 94, revealing the karyotype formula to be 2n=16m+28sm+4st+2t, NF=94. Regarding the comparison of mitochondrial 16S rRNA genes of the 14 Barbidae species, the genetic distances ranged from 0.016 to 0.134, with an average of 0.074. Notably, the interspecific genetic distances between L. hoevenii and Onychostoma barbata was the largest at 0.134, while that between L. hoevenii and Sikukia stejnegeri was the smallest at 0.090. Finally, the resulting phylogenetic tree showed that these 14 species were clustered into two respective branches, indicating a closer relationship between L. hoevenii and Barbodes hexagonlepis. L. hoevenii was basal in the phylogenetic tree. These results provide a foundation for the evaluation, breeding, and phyletic evolution of fish germplasm resources.