Twenty-one novel polymorphic microsatellite loci were isolated from the Chinese soft-shelled turtle, Pelodiscus sinensis (Trionychidae). This is a commonly consumed Asian species, especially in China, where it is reared in large numbers on farms. We screened 34 unrelated individuals from Wuhu of Anhui province in China and detected high levels of polymorphism for all 21 loci, with the number of alleles/locus ranging from 5 to 23 (mean 15.67). The expected and observed heterozygosities ranged from 0.421 to 0.946 and from 0.324 to 0.941, respectively. Hence, these microsatellites could facilitate studies on genetic diversity and population structure and marker-assisted breeding of this vulnerable species.