The Pelteobagrus vachelli and Leiocassis longirostris, belong to the order Siluriformes and family Bagridae, respectively fall under the genus Pelteobagrus and Leiocassis. Their interspecific hybrid offspring exhibit superior growth performance, suggesting significant production potential. As the hybrid offspring of P. vachelli and L. longirostris share similar morphological features with their parents, confusion may arise in production. DNA molecular markers can be used to accurately and rapidly distinguish species, and are commonly used for species identification. In this study, based on the published genomic data of P. vachelli and L. longirostris, comparative genomic analysis revealed a close evolutionary relationship between the two species. The estimated divergence time was about 3.7 million years ago. Moreover, through chromosome sequence synteny analysis, it was found that both species had 26 pairs of chromosomes. These chromosomes corresponded one-to-one, with no observed chromosomal fusions, only intra-chromosomal rearrangements. Based on these results, a 391 bp INDEL was identified in the patj gene sequence in the two fish species. Using this differential fragment, we designed the primer PVLL. This primer only amplified a 339 bp band in P. vachelli and a 730 bp band in L. longirostris, while in the hybrid species, it amplified both 339 bp and 730 bp bands. The difference in the amplified bands could be used to distinguish the three species, regardless of gender accurately . We further verified the validity of the primers in different populations. Accordingly, our study provides a theoretical basis for hybrid breeding between P. vachelli and L. longirostris through comparative genomic analysis. Furthermore, we developed a molecular marker that could efficiently and accurately identify P. vachelli, L. longirostris, and their interspecific hybrids, which will greatly improve the efficiency of hybrid breeding production.