Darkbarbel catfish (Pelteobagrus vachelli) is an important commercial fish species in China. However, its wild populations have undergone a dramatic decline in recent years because of overfishing and habitat destruction for its spawning. Therefore, it is extremely urgent to develop sufficient molecular markers for the effective conservation and molecular-aided selection of this species. In the present study, 24 polymorphic single nucleotide polymorphism (SNP) markers were developed using restriction-site associated DNA tags sequencing (RAD-seq) in P. vachelli. Genetic diversity analysis revealed that the observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.190 to 0.900 and 0.251 to 0.508, respectively, and the polymorphism informatin content (PIC) ranged from 0.215 to 0.375. Among these SNPs, 3 loci showed significant departures from Hardy–Weinberg equilibrium after Bonferroni adjustments. The panel of polymorphic SNPs developed here will be useful for genetic conservation and selective breeding in darkbarbel catfish.