The narrow genetic base of the cultivated tomato, Solanum lycopersicum L., necessitates introgression of new variation from related species. Wild tomato species represent a rich source of useful genes and traits. Exploitation of genetic variation within wild species can be facilitated by the use of molecular markers and genetic maps. Recently we identified an accession (LA2093) within the red-fruited wild tomato species Solanum pimpinellifolium L. with exceptionally desirable characteristics, including disease resistance, abiotic stress tolerance, and high fruit lycopene content. To facilitate genetic characterization of such traits and their exploitation in tomato crop improvement, we developed a new recombinant inbred line (RIL) population from a cross between LA2093 and an advanced tomato breeding line (NCEBR-1). Furthermore, we constructed a medium-density molecular linkage map of this population using 294 polymorphic markers, including standard RFLPs, EST sequences (used as RFLP probes), CAPS, and SSRs. The map spanned 1091 cM of the tomato genome with an average marker spacing of 3.7 cM. A majority of the EST sequences, which were mainly chosen based on the putative role of their unigenes in disease resistance, defense-related response, or fruit quality, were mapped onto the tomato chromosomes for the first time. Co-localizations of relevant EST sequences with known disease resistance genes in tomato were also examined. This map will facilitate identification, genetic exploitation, and positional cloning of important genes or quantitative trait loci in LA2093. It also will allow the elucidation of the molecular mechanism(s) underlying important traits segregating in the RIL population. The map may further facilitate characterization and exploitation of genetic variation in other S. pimpinellifolium accessions as well as in modern cultivars of tomato.