The Chinese longsnout catfish (Leiocassis longirostris Günther) is a semi-migratory freshwater species. Leiocassis longirostris is mainly inhabiting the main stream of the Yangtze River and rarely found in lakes due to over fishing, environmental pollution and other human disturbances. Novel single nucleotide polymorphisms (SNPs) were screened based on high-throughput sequencing in Leiocassis longirostris. In this study, 40 SNP markers were developed successfully with 30 individuals from the Yangtze River segment of Nanjing, China. The observed heterozygosity (Ho) ranged from 0.0003 to 1.0000, while the expected heterozygosity (He) varied from 0.3367 to 0.9390. The minor allele frequency ranged from 0.0167 to 0.2500. Thirty-four SNPs have been confirmed to fit Hardy–Weinberg equilibrium after Bonferroni correction (P < 0.0013). The first set of SNP makers developed from Leiocassis longirostris will provide valuable informating in population genetics and resource conservation.