Whiteflies [Bemisia tabaci (Gennadius)] and aphids [Aphis gossypii Glover and Myzus persicae (Sulzer)] are serious threats to watermelon by direct feeding and by transmitting viruses of important virus diseases. The desert watermelon Citrullus colocynthis (L.) has been shown to exhibit resistance to these insect pests and could be a useful source for breeding resistance into watermelon [Citrullus lanatus var. lanatus (Thunbs) Matsum & Nakai]. Using high-performance liquid chromatography (HPLC), we found differences among the chemical profiles of two U.S. PIs of C. colocynthis, one PI of C. lanatus var. citroides, and two heirloom watermelon (C. lanatus var. lanatus) cultivars (‘Charleston Gray’ and ‘Mickey Lee’). Flavonoid and caffeic acid derivatives were identified in the leaf extracts by a combination of ultraviolet (UV) and mass spectrometry (MS) spectral analyses. Four phenolic derivatives of caffeic and/or ferulic acid were found to be essentially unique to C. colocynthis. Total flavonoid content was found to be approximately four to 18 times higher in C. colocynthis accessions and seven to nine times higher in C. lanatus var. citroides as compared with watermelon cultivars. Caffeoyl-glucose was also identified in the leaves of watermelon cultivars for the first time. Leaf sugar concentrations (198 to 211 mg·dL−1), read from a glucometer, were statistically the same among the various germplasm entries. These results will help in the development of pest-resistant watermelon.