The Chinese grass shrimp, Palaemonetes sinensis, is an economically important freshwater shrimp in China, and the study of genetic diversity and structure can positively contribute to the exploration of germplasm resources and assist in the understanding of P. sinensis aquaculture. Microsatellite markers are widely used in research of genetic backgrounds since it is considered an important molecular marker for the analyses of genetic diversity and structure. Hence, the aim of this study was to evaluate the genetic diversity and structure of wild P. sinensis populations in China using the polymorphic microsatellite makers from the transcriptome. Sixteen polymorphic microsatellite markers were developed for P. sinensis from transcriptome, and analyzed for differences in genetic diversity and structure in multiple wild P. sinensis populations in China. Totally of 319 individual shrimps from seven different populations were genotyped to find that allelic polymorphisms varied in two to thirteen alleles seen in the entire loci. Compared to other populations analyzed, the two populations including LD and SJ showed lower genetic diversity. Both the genetic distance (D) and Wrights fixation index (F ) comparing any two populations also indicated that LD and SJ populations differed from the other five populations. An UPGMA tree analysis showed three main clusters containing SJ, LD and other populations which were also confirmed using STRUCTURE analysis. This is the first study where polymorphic microsatellite markers from the transcriptome were used to analyze genetic diversity and structures of different wild P. sinensis populations. All the polymorphic microsatellite makers are believed useful for evaluating the extent of the genetic diversity and population structure of P. sinensis. Compared to the other five populations, the LD and SJ populations exhibited lower genetic diversity, and the genetic structure was differed from the other five populations. Therefore, they needed to be protected against further declines in genetic diversity. The other five populations, LP, LA, LSL, LSY and LSH, are all belonging to Liaohe River Drainage with a relatively high genetic diversity, and hence can be considered as hot spots for in-situ conservation of P. sinensis as well as sources of desirable alleles for breeding values.