The dojo loach Misgurnus anguillicaudatus is an endemic freshwater species to Asia. The effective conservation and molecular-aided selection of M. anguillicaudatus have been limited without sufficient molecular markers. In this study, 112 novel single nucleotide polymorphisms (SNPs) were screened based on 2b-RAD sequencing database, and 57 SNP markers were developed and characterized by genotyping 40 individuals using SNaPshot method. The observed heterozygosity (Ho) ranged from 0.025 to 0.675, while the expected heterozygosity (He) varied from 0.025 to 0.500. The minor allele frequency (MAF) ranged from 0.013 to 0.500. Among these SNPs, 18 loci were found to deviate significantly from the Hardy–Weinberg equilibrium after Bonferroni correction (P < 0.05). The first set of SNP markers developed from M. anguillicaudatus will provide valuable information in further population genetic analysis and natural resource conservation.