Tomato and potato expressed sequence tag (EST) sequences contained in the solanaceae genomics network (SGN) database were screened for simple sequence repeat (SSR) motifs. A total of 609 SSRs were identified and assayed on Solanum lycopersicum LA925 (formerly Lycopersicon esculentum) and S. pennellii LA716 (formerly L. pennellii). The SSRs that did not amplify, gave multiple band products, or did not exhibit a polymorphism that could be readily detected on standard agarose gels in either of these species were eliminated. A set of 76 SSRs meeting these criteria was then placed on the S. lycopersicum (LA925) x S. pennellii (LA716) high-density map. A set of 76 selected cleaved amplified polymorphism (CAP) markers was also developed and mapped onto the same population. These 152 PCR-based anchor markers are uniformly distributed and encompass 95% of the genome with an average spacing of 10.0 cM. These PCR-based markers were further used to characterize S. pennellii introgression lines (Eshed and Zamir, Genetics 141:1147-1162, 1995) and should prove helpful in utilizing these stocks for high-resolution mapping experiments. The majority of these anchor markers also exhibit polymorphism between S. lycopersicum and two wild species commonly used as parents for mapping experiments, S. pimpinellifolium (formerly L. pimpinellifolium) and S. habrochaites (formerly L. hirsutum), indicating that they will be useful for mapping in other interspecific populations. Sixty of the mapped SSRs plus another 49 microsatellites were tested for polymorphism in seven tomato cultivars, four S. lycopersicum var. cerasiforme accessions and eight accessions of five different wild tomato species. Polymorphism information content values were highest among the wild accessions, with as many as 13 alleles detected per locus over all accessions. Most of the SSRs (90%) had accession-specific alleles, with the most unique alleles and heterozygotes usually found in accessions of self-incompatible species. The markers should be a useful resource for qualitative and quantitative trait mapping, marker-assisted selection, germplasm identification, and genetic diversity studies in tomato. The genetic map and marker information can be found on SGN (http://www.sgn.cornell.edu).