Abstract: To explore the genetic diversiy and diffentation degree of Pseudolaubuca engraulis population in the Pearl River Basin, RAD-Seq technology was used to develop microsatellite loci. A total of 100 pairs of microsatellite primers were designed, 66 of which could stably amplifiy the target band, including 16 polymorphic microsatellite markers (PIC>0.5) which were mostly with 2 base repeats. Ten polymorphic microsatellite loci were used in genetic diversity analysis in five P. sinensis populations of Pearl River Basin (YuJ, ZJ, YJ, RJ, GJ). The effective allele number, observed heterozygosity, and expected heterozygosity varied from 5.2028—6.3800, 0.6773—0.7667, and 0.7975—0.8425, respectively. The results showed that five P. sinensis populations in the Pearl River Basin had a high level of genetic diversity level, the YJ population had the highest genetic diversity while the RJ population had the lowest. The genetic parameters of population differed little, indicating that their genetic diversity level was close. AMOVE analysis showed that the genetic variation mainly come from population (98.45%), and only a small part of the variation came from different population (1.55%). The genetic differentiation coefficient F_st of the total population was 0.015, which was a low degree of differentiation. The gene exchange value (N_m) was between 8.2246 and 64.3526. The results showed that there were frequent gene exchange in different populations and strong resistance to genetic drift. This study screened microsatellite loci with high polymorphism and used the loci to evaluate the genetic diversity of the P. sinensis population, aiming to effectively monitor the germplasm resources of P. sinensis and to provide scientific guidance for its resource development, utilization and protection.