The largemouth bass Micropterus salmoides was introduced into Guandong province of China in 1983, and is now one of the China’s primary cultured fish species. After a long period of farming, the populations are facing the stringent challenges of germplasm resources degraded, and genetic improvement is urgently needed. Thus, developing sufficient molecular markers for the conservation and molecular-aided selection of largemouth bass is urgent. In this study, we implemented restriction site associated DNA sequencing (RAD-seq) to develop single nucleotide polymorphism (SNP) markers. In total, 40 novel SNP markers were found to be polymorphic and showed bi-allelic in 49 individuals of largemouth bass. Those novel SNP markers may be useful for further genetic improvement of largemouth bass.