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The bitter desert watermelon, Citrullus colocynthis (L.) Schrad is a wild species valuable for biotic and abiotic stress resistance that could be exploited for improving watermelon cultivars [Citrullus lanatus (Thunberg) Matsum & Nakai var. lanatus]. The objective of this study was to survey and identify C. colocynthis accessions displaying resistance to the Papaya ringspot virus-watermelon strain (PRSV-W). Thirty-one accessions of C. colocynthis, collected in Africa, the Middle East, southwest Asia, and India were evaluated for PRSV-W resistance. Of these 31 accessions, 4 U.S. Plant Introduction (PI) accessions, including 525080 (collected in Qena, Egypt) and PI 537277, PI 652554, and Griffin 14201 (collected at the northern Indian desert of Rajasthan and the neighboring region of Punjab, Pakistan) showed high resistance to PRSV-W. Plants of these four resistant PIs were self-pollinated to produce S1 and S2 seeds that continued to maintain the high levels of PRSV resistance. Since there is a wide genetic distance between watermelon cultivars and C. colocynthis, we performed crosses and backcrosses with watermelon cultivars, including ’Charleston Gray’ and ’sugar Baby’ to produce viable seed that would be useful in the development of genetic populations and in introducing the resistance into watermelon cultivars.