Producers of fresh fruits and vegetables face increasing production costs and international market competition. Growers who can offer high-quality watermelons [Citrullus lanatus (Thumb.) Matsum. &; Nakai] that are also highly nutritious will have better market opportunities. To accomplish that, germplasm must be identified that has enhanced phytonutrient levels. Surprisingly, there is little information on the genetics of nutritional quality in watermelon. The present study was performed on 56 watermelon cultivars, breeding lines, and PI accessions (hereafter collectively referred to as cultigens) to determine the importance of genotype and environmental effects on L-citrulline concentration in fruit, an amino acid that helps regulate blood pressure. Our results demonstrated that L-citrulline concentration was affected by environment and the amount of environmental effect varies among cultigens. The mean of fruit tested in Lane, OK, was 3.10 mg·g-1 fresh weight and in College Station, TX, it was 1.67 mg·g-1 fresh weight. All cultigens had a higher mean L-citrulline concentration when grown in Lane, OK, instead of College Station, TX. Additionally, the L-citrulline concentration varied considerably within cultigens; i.e., 'Congo' had a 1.26 to 7.21 mg·g-1 fresh sample deviation. The cultigen 'AU-Jubilant' had the most stable L-citrulline concentration (2.23 to 4.03 mg·g-1 fresh deviation) when tested from one location. Environment did not significantly increase within-genotype variation (average se of 10 cultigens tested at each location was ± 35.3% for College Station, TX, and ± 32.9% for Lane, OK). L-citrulline concentration did not correlate with watermelon type (open-pollinated or F1 hybrid) or flesh color (red, orange, salmon yellow, or white). Differences among cultigens for L-citrulline were large (1.09 to 4.52 mg·g-1 fresh sample). The cultigens with the highest L-citrulline concentration were 'Tom Watson', PI 306364, and 'Jubilee'. These could be used to develop cultivars having a high concentration of L-citrulline.