BACKGROUND: The development of genetic markers is crucial for aquaculture and population genetics. Polyallelic microsatellite markers are widely used for breeding and family management of Eriocheir sinensis (E. sinensis), contributing to the sustainable development of the industry. METHODS: Through transcriptome analysis, 100 highly polymorphic SSR markers were identified and developed. From these, four multiplex PCR panels, each containing 16 markers, were constructed and applied to parentage assignment in six E. sinensis families. RESULTS: The number of alleles (N(a)) ranged from 7 to 21, the observed heterozygosity (H(o)) varied from 0.356 to 0.951, and the expected heterozygosity (H(e)) varied from 0.656 to 0.909. The polymorphism information content (PIC) ranged from 0.633 to 0.900. The CERVUS analysis showed that using only the two most informative multiplex PCR panels, assignment success rates exceeded 90.34%, with 100% accuracy in assigning offspring to parents when three or four panels were used. CONCLUSIONS: This method significantly enhances the efficiency of microsatellite marker analysis, which provides a convenient and cost-effective method for breeding programs and family management of E. sinensis.