The evaluation of environmental and genotypic effects on fruit physical and chemical characteristics enables assessment of the feasibility of selecting diploid and/or triploid cultivars for either specific or more diverse locations. Isolation and characterization of genes encoding enzymes in the carotenoid biosynthetic pathway provides fundamental genetic information which can facilitate breeding of watermelon cultivars having desirable flesh colors and enhanced beneficial carotenoids. For the environmental studies, the effects of deficit irrigation on lycopene content, total soluble solids, firmness, and yield of diploid and triploid watermelon were evaluated in different locations and growing seasons. Irrigation regimes were 1.0 evapotranspiration (ET), 0.75 ET, and 0.5 ET. To investigate if there is a consistent response in cultivars across diverse locations, studies were conducted in three distinct Texas regions. Deficit irrigation reduced total marketable yield, and increased the yield of small fruits. Location and irrigation regimes had major influences on yield. Soluble solids content increased with deficit irrigation at 0.5 ET in triploids, but not in diploids. Flesh firmness also increased in triploids compared to diploids. Lycopene content increased with maturity at all irrigation regimes and cultivars. This work confirms that deficit irrigation directly reduces yield, but does not reduce lycopene and fruit quality of the triploids used in this study. From the genetic studies, a total of eight genes encoding enzymes in the carotenoid biosynthetic pathway were isolated and characterized. Two members of the phytoene synthase (PSY) gene family were identified; PSY-A was expressed in all type of tissues, but PSY-B transcript was detected only in ovary, leaf, and root tissues. Gene expression of carotenoid isomerase (CRTISO) was not detected in salmon yellow. A color inheritance study of watermelon flesh indicated that a single gene might determine color difference between canary yellow and red without an inhibitory effect. A cleaved amplified polymorphic sequence (CAPS) marker developed from the SNP marker tagging two different lycopene β-cyclase (LCYB) alleles cosegregated perfectly with color phenotypes. It was concluded that color determination may be due to a reduced activity of LCYB enzyme in red, whereby a phenylalanine is conserved among canary yellow and valine is conserved among red watermelon.