A large number of polymorphic genetic markers are needed to examine genetic variation in wild and cultured penaeid species, trace pedigrees, and apply marker-assisted selection in breeding programs. The objectives of this study are to (1) isolate and characterize microsatellite genetic markers for the Pacific whiteleg shrimp, Litopenaeus vannamei, (2) demonstrate the usefulness of three randomly selected markers to examine allelic variation in wild and cultured shrimp populations, and trace the pedigree of two families from the breeding program of the US Marine Shrimp Farming Program (USMSFP); and (3) determine the potential usefulness of these microsatellites for linkage mapping. A total of 128 recombinant clones obtained from Sau3 A-digested genomic libraries prepared from ovary of specific pathogen-free L. vannamei were sequenced; 86 of which contained simple sequence repeats (SSRs), or microsatellites, with three or more repeat motifs. The frequency of microsatellites with five or more repeats was estimated at 1/2.74 kb. The most abundant di-, tri, tetra-, penta-, and hexa-nucleotide motifs were (CT)n, (CCT)n and (CTT)n, (CATA)n, (CTTCT)n, and (GAGATA)n. The octa-nucleotide (CCCTCTCT)3 was also identified. Sixty-two primer sets flanking microsatellites with single or multiple motifs were designed and tested for polymorphism with a small test panel representing individuals of the mapping families being used to develop a linkage map for L. vannamei (ShrimpMap), and 35 of these (56.4%) were polymorphic. Three of these markers (TUGAPv1-3.224, TUGAPv5-7.33, and TUGAPv7-9.17) were used for estimating allele diversity of wild populations of Ecuador and Mexico and tracing the pedigree in two families of the USMSFP breeding program. A large number of alleles (21–31) and allele size range (95–275 bp) was observed in wild shrimp. There was a large allele size range difference at all three loci examined, being smaller in cultured shrimp (32–74 bp) than in wild shrimp (77–180 bp), suggesting null alleles or mutations. The presence of stuttering bands with marker TUGAPv5-7.33 made it difficult to score the wild shrimp from Mexico and suggest the need to first test for inheritance pattern of shrimp microsatellites before using them in population genetics, relatedness/kinship, and traceability studies. Allele segregation in cultured shrimp confirmed codominant inheritance of markers. Observed heterozygosity was 100% for all loci scored. Fourteen randomly selected polymorphic markers were further genotyped with the entire IRMF panel and 8 of these amplified with most of the individuals tested. Linkage analysis using CRIMAP with LOD score of 5.0 placed four of the markers (TUGAPv1-3.132, TUGAPv3-5.213, TUGAPv7-9.179, and TUGAPv7-9.95) in linkage groups LG6, LG5, LG13, and LG14, respectively, and four markers (TUGAPv3-5.271, TUGAPv3-5.391, TUGAPv7-9.94, and TUGAPv7-9.226) remained unlinked. In summary, 35 new microsatellites were developed for L. vannamei, some of which are useful for studies on genetic diversity of wild and cultured stocks, pedigree tracing in breeding programs, and linkage mapping. Moreover, some of the genomic sequences reported here had significant homology to hypothetical proteins of various organisms, known (e.g., reverse transcriptase) or unknown genes, or no homology to any sequence in the GeneBank database, suggesting that sequences from a genomic library can also provide valuable information in identifying functional markers in shrimp.