Technical Abstract: In this study, we report a simple procedure for developing gene targeted primers, named high-frequency gene sequence (HFGS) primers, based on oligonucleotides that exist in high frequency in watermelon expressed sequence tag (EST)-unigenes. These HFGS primers were constructed by first screening for 8, 9, and 10 base sequences that exist in high frequency in 4,700 watermelon fruit EST-unigenes [International Cucurbit Genomics Initiative (ICuGI); http://www.icugi.org]. The screening, using an in house Perl script, yielded 3,200 oligonucleotides that exist 32 to 335 times in the 4,700 EST-unigens. Then, one-hundred and ninety-two HFGS oligonucleotide (87.5-100% GC content) were used for primer construction. The 192 HFGS-primers were tested in PCR reactions with genomic DNA of the watermelon cultivars Allsweet, Crimson Sweet, Charleston Gray and Dixie Lee, known to have close genetic similarity, based on analysis with 85 RAPD primers. The average number of bands produced by the HFGS primers among these four watermelon cultivars was comparable (5.1 bands per primer) to the number of bands produced by RAPD primers (5.6 bands per primer). However, the HFGS primers produced a higher number of polymorphic bands (an average of 1.77 bands per primer) than RAPD primers (an average of 0.73 polymorphic bands per primer). Because they are derived from EST-unigenes, the HFGS primers may reveal new genomic loci and be more useful in gene tagging and genetic mapping than RAPD primers.