Technical Abstract: A novel watermelon breeding line was developed at the USDA, ARS, U.S. Vegetable Laboratory, Charleston, SC 29414. This breeding line contains the nuclear genome of cultivated watermelon (Citrullus lanatus var. lanatus) and the chloroplast and mitochondrial genomic background of the desert species C. colocynthis. USVL-220 is an ‘Allsweet’-type watermelon, with an elongated fruit (16.00” long and 7.5” width) and a light green dappled rind (1-1.2” thick). In field plots at Charleston, SC (2004-2005), USVL-220 plants produced one female flower for every 7-8 male flowers, and 2.6-3 mature fruits (12-14 lb per fruit). A typical fruit has red color flesh with slightly crispy texture (8-8.5% solid soluble content), and does not exhibit hollow heart. They are ready for harvest in mid-late season (80 days post planting). USVL-220 might be useful for scientists and plant breeders interested in studying effects of foreign cytoplasm (chloroplast and mitochondria) on photosynthesis, respiration, disease or pest resistance, flower production, and fruit quality in cultivated watermelon. USVL-220 might be useful in breeding programs to enhance rind color (light green dappled) and thickness (1-1.2"), flesh color and texture, and fruits devoid of hollow heart.