Phytophthora fruit rot, caused by Phytophthora capsici, is prevalent in most watermelon-producing regions of southeastern United States and is known to cause pre- and post-harvest yield losses. A non-wound inoculation technique was developed to evaluate detached mature fruit belonging to U.S. watermelon PIs for resistance to fruit rot caused by P. capsici. Mature fruit were harvested and placed on wire shelves in a walk-in humid chamber [greater than 95% relative humidity (RH), temperature 26 ± 2 °C] and inoculated with a 7-mm agar plug from an actively growing colony of P. capsici. Twenty-four PIs that exhibited resistance in a preliminary evaluation of 205 PIs belonging to the watermelon core collection in 2009 were grown in the field and greenhouse in 2010 and 2011 and evaluated in the walk-in humid chamber. Fruit rot development was rapid on fruit of susceptible controls ‘Black Diamond’, ‘Sugar Baby’, and PI 536464. Several accessions including PI 560020, PI 306782, PI 186489, and PI 595203 (all Citrullus lanatus var. lanatus) were highly resistant to fruit rot. One C. colocynthis (PI 388770) and a C. lanatus var. citroides PI (PI 189225) also showed fruit rot resistance. Fruit from PIs that were resistant also had significantly lower amounts of P. capsici DNA/gram of fruit tissue compared with the susceptible commercial cultivars Sugar Baby and Black Diamond. The sources of resistance to Phytophthora fruit rot identified in this study may prove useful in watermelon breeding programs aimed at enhancing disease resistance.