Mauremys reevesii and M. sinensis are economically important freshwater turtle species widely distributed in China. However, due to heavy overexploitation, their wild populations are near extinction and most living resources are no intensively cultured in breeding farms. To set up an effective management and conservation strategy for both species, we carried out extensive restriction site-associated DNA sequencing (RAD-seq) to develop robust single-nucelotide polymorphism (SNP) markers. A total of 144 novel high-quality SNP markers were isolated and characterized from four populations in China, including Anhui P.R., Guangdong P.R., Hubei P.R. and Taiwan P.R., five of each population. The values of observed and expected heterozygosity ranged respectively from 0.0000 to 0.7895 and from 0.0950 to 0.5000. The inbreeding coefficient varied from − 0.6087 to 1.0000, and 79 SNPs significantly deviated from Hardy–Weinberg equilibrium (P < 0.05). These novel markers will facilitate future studies on the population and conservation of M. reevesii and M. sinensis.