The Pacific white shrimp, Litopenaeus vannamei, is one of the most economically important marine aquaculture species in China, as well as the world. A large number of molecular markers are required for the studies of genetic diversity and breeding in shrimp. In the present investigation, 14767 SSRs were detected in L. vannamei unigenes derived from transcriptome sequencing. Among these, dinucleotide SSRs were the most abundant motif (59.53%), followed by trinucleotide (35.78%) SSRs. A total of 74 primer pairs were designed and validated using DNA pools. 54 pairs (72.97%) were amplified with clear band, and 27 pairs (36.49%) were found to be polymorphic. The genetic diversity was analyzed using 11 highly polymorphic SSR markers in the breeding population of "Kehai No.1, " a selected new variety of L. vannamei. Consequently, the allele number of these microsatellites ranged from 2 to 11. Expected heterozygosity and observed heterozygosity ranged from 0.256 to 0.858 and 0.213 to 0.875, re-spectively. The polymorphic information varied from 0.221 to 0.830. Four loci deviated significantly from Hardy-Weinberg equilibrium (HWE) (P < 0.05). The large number of genetic markers developed in the present study would be highly beneficial to the researches to understand genetic diversity, selective breeding, and germ-plasm conservation in shrimp.