Despite the collection and availability of abundant tomato genome sequences, PCR-based markers adapted to large scale analysis have not been developed in tomato species. Therefore, using public genome sequence data in tomato, we developed three types of DNA markers: expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers (TES markers), genome-derived SSR markers (TGS markers) and EST-derived intronic polymorphism markers (TEI markers). A total of 2,047 TES, 3,510 TGS and 674 TEI markers were established and used in the polymorphic analysis of a cultivated tomato (Solanum lycopersicum) 'LA925' and its wild relative Solanum pennellii 'LA716', parents of the Tomato-EXPEN 2000 mapping population. The polymorphic ratios between parents revealed by the TES, TGS and TEI markers were 37.3, 22.6 and 80.0%, respectively. Those showing polymorphisms were used to genotype the Tomato-EXPEN 2000 mapping population, and a high-density genetic linkage map composed of 1,433 new and 683 existing marker loci was constructed on 12 chromosomes, covering 1,503.1 cM. In the present map, 48% of the mapped TGS loci were located within heterochromatic regions, while 18 and 21% of TES and TEI loci, respectively, were located in heterochromatin. The large number of SSR and SNP markers developed in this study provide easily handling genomic tools for molecular breeding in tomato. Information on the DNA markers developed in this study is available at http://www.kazusa.or.jp/tomato/.