A preliminary low-density linkage map (ShrimpMap) based solely on simple sequence repeats (SSRs) or microsatellite genetic markers was constructed for specific pathogen-free (SPF) shrimp, Litopenaeus vannamei, from the United States Marine Shrimp Farming Program (USMSFP). Marker loci originated mostly from genomic libraries cloned using DNA from ovaries of adult shrimp or cDNA libraries cloned using RNA of juveniles challenged with either Taura syndrome virus (TSV) or white spot syndrome virus (WSSV). Out of approximately 150 polymorphic markers initially tested with a small test panel consisting of 16 individuals, 100 (83 genomic SSRs and 17 EST-SSRs) were useful for genotyping with the entire mapping panel, a three-generation pedigree comprising two grandparents, two parents and 89 progeny. High frequency of null alleles (approx. 25% of markers) was observed in shrimp microsatellites. Chi-square goodness-of-fit tests revealed that 30 of the 100 markers showed segregation distortion, 17 of which were highly significant distorted (P < 0.001). Linkage analysis using CRIMAP with a LOD score of 3.0 provided a map with coverage for 14 linkage groups consisting of 67 linked markers spaced every ∼22 cM with an observed genome length of 689.1 cM and an estimated genome length for L. vannamei of 2675.7 cM. Variable results were obtained using a LOD score of 5.0. Sex-linked microsatellites were identified in linkage group 4 (LG4) of ShrimpMap, a linkage group that also includes 28 s rRNA, nonLTR retrotransposon reverse transcriptase and other markers with no homology to any sequence in the GenBank database. The map includes the first EST-SSR markers mapped for L. vannamei. Human ESTs also amplified in shrimp DNA and two of them were useful for linkage analysis. Work is underway to place additional EST-SSR markers on to ShrimpMap to serve as a framework map for penaeid species to facilitate integration of linkage and physical maps, study conservation and evolutionary genomics in penaeids, and search for candidate genes associated with fitness traits in wild and cultured shrimp. Moreover, the mapped EST-SSRs provide valuable comparative genomic links not only between L. vannamei and other penaeid species but also between shrimp and other invertebrate and vertebrate genomes.