Understanding the natural mating behavior (self- or cross-pollination) in watermelon is important to the design of a suitable breeding strategy. The objective of this study was to measure the rate of self- and cross-pollination in watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] using the dominant gene Sp (Spotted leaves and fruit) as a marker. The experiment consisted of two studies and was a split plot in a randomized complete block design with 3 years (2009 to 2011) and four locations (Clinton, Kinston, Oxford, Lewiston, NC). For the intercrossing study, whole plots were the two spacings (1.2 × 0.3 m and 1.2 × 0.6 m) with four replications in 2010. For the inbreeding study, whole plots were two equidistant spacings (3 × 3 m and 6 × 6 m) with four replications in 2009 to 2011. Cultivars Allsweet and Mickylee were subplots within each whole plot. In the inbreeding study, spacing and year had a significant effect on the rate of self-pollination, which was moderate (47% and 54%, respectively) when watermelon plants were trained in a spiral and spaced 3 × 3 m or 6 × 6 m apart. Spacing and cultivar did not have a significant effect on cross-pollination in the intercrossing study. Closely spaced watermelon plants (1.2 × 0.3 m and 1.2 × 0.6 m) had low natural outcrossing rate (31% and 35%, respectively) and was not adequate to intercross families. However, breeders should consider the amount of self-pollination in watermelon to calculate the estimates of component of genetic variances.