A normalized cDNA library was constructed using watermelon flesh mRNA from three distinct developmental time-points and was subtracted by hybridization with leaf cDNA. Random cDNA clones of the watermelon flesh subtraction library were sequenced from the 5′ end in order to identify potentially informative genes associated with fruit setting, development, and ripening. One-thousand and forty-six 5′-end sequences (expressed sequence tags; ESTs) were assembled into 832 non-redundant sequences, designated as “EST-unigenes”. Of these 832 “EST-unigenes”, 254 (∼30%) have no significant homology to sequences published so far for other plant species. Additionally, 168 “EST-unigenes” (∼20%) correspond to genes with unknown function, whereas 410 “EST-unigenes” (∼50%) correspond to genes with known function in other plant species. These “EST-unigenes” are mainly associated with metabolism, membrane transport, cytoskeleton synthesis and structure, cell wall formation and cell division, signal transduction, nucleic acid binding and transcription factors, defense and stress response, and secondary metabolism. This study provides the scientific community with novel genetic information for watermelon as well as an expanded pool of genes associated with fruit development in watermelon. These genes will be useful targets in future genetic and functional genomic studies of watermelon and its development.