Gene identification and characterization can be utilized for the identification of respective functions and their relationship to flesh color inheritance. Phytoene synthase (PSY), which converts two molecules of GGPP into phytoene, is the first committed step of the pathway. Previous phylogenetic analysis of PSY has indicated that PSY duplication is common in Poaceae, but rare in dicots. Degenerate PCR and RACE were used for PSY cloning. Three members of PSY gene family (PSY-A, PSY-B and PSY-C) were identified. PSY-A shared higher identity with PSY-C than PSY-B. PSYC shared 96% identity with melon PSY. PSY-C also showed a high homology with tomato PSY1, even higher than PSY-A and PSY-B. It showed a similar gene expression pattern, so we propose that PSY-C is a homologue to PSY1. RT-PCR analysis indicated that PSY-B has a different transcriptional behavior from PSY-A, similar to tomato PSY2. Therefore, PSY genes appear to be under different regulatory mechanisms. Deduced protein sequence of PSY1 or PSY2 between species has higher homology than between PSY1 and PSY2 within species. Phylogenetic analysis indicated that watermelon PSY gene family is very distantly related. Watermelon and carrot PSY gene families did not appear to cluster as closely as in Poaceae or tomato. This indicates that watermelon and carrot PSY genes are not conserved as much as PSY in tomato or Poaceae. There was no particular pattern in phylogenetic relationship of dicots. Poaceae PSY genes showed a clustering into a PSY1 group and PSY2 group. PSY duplication in watermelon provides additional evidence that PSY duplication may be a common phenomenon in dicots. They are likely to be duplicated evolutionarily a long time ago, possibly even prior to the evolution of monocot and dicot divergence.