Largemouth bass (Micropterus salmoides) is an economically important species in China. Contrary to its rapidly increasing yield during the last decades, the domestic genetic diversity of largemouth bass has gradually declined. In this study, 37 single nucleotide polymorphism (SNP) markers were developed based on genotyping-by-sequencing data and characterized by genotyping 32 individuals using the PCR–restriction fragment length polymorphism method. The effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphic information content (PIC) of these SNPs ranged from 1.168 to 1.998, 0.156 to 0.844, 0.146 to 0.507, and 0.134 to 0.375, respectively. Totally, five loci deviated significantly from Hardy–Weinberg equilibrium (p < 0.05), while there existed no linkage disequilibrium at all loci. These novel polymorphic markers will lay the foundation for future rationally excavation and utilization of germplasm resources in M. salmoides.