A total of 1479 recombinant clones were obtained from a Sau3A-digested genomic library of Penaeus (Litopenaeus) vannamei and used for probe hybridization. Of the 251 clones that tested positive to one or more of the probes and were sequenced, 173 (69%) contained 573 simple sequence repeats, or microsatellites, with 3 or more repeats. The frequency of microsatellites with 3, 5, and 10 or more repeats was 1 in 0.94 kb, 1 in 2.78 kb, and 1 in 5.94 kb, respectively. To increase the number of polymorphic markers for mapping, 136 primer sets that flanked microsatellites containing single or multiple motifs with 3 or more repeats were designed and tested. Of the 136 primers, 93 (68.0%) were polymorphic in cultured shrimp, with polymorphism information content (PIC) values ranging from 0.195 to 0.873, and observed heterozygosities ranging from 10% to 100%. These markers are being used along with other markers to construct a linkage map for P. vannamei.