Leiobagrus kingi is a species endemic to southwest China, it is listed as an endangered species. Due to habitat destruction, over-fishing and water pollution, the wild population of L. kingi has decreased dramatically in recent years. Insufficient molecular markers have limited the effective conservation of this species. In the present study, 35 novel single-nucleotide polymorphism (SNP) markers in L. kingi were isolated and characterized based on restriction site-associated DNA sequencing (RAD-seq). The minor allele frequency ranged from 0.043 to 0.481, and the observed and expected heterozygosities ranged from 0.077 to 0.678 and 0.105 to 0.505, respectively. Polymorphic information content ranged from 0.072 to 0.411. Among these SNPs, five loci showed significant departures from the Hardy–Weinberg equilibrium (P < 0.01). The novel polymorphic SNPs will be useful for future investigation of population structure and conservation genetics for this species.