Knowledge of relative genetic distance among genotypes is useful in a breeding program because it permits organization of germplasm resources. Genetic distance (GD) was estimated among 65 Phaselous lunatus L.. accessions, which included 4 large-seeded and 7 small-seeded cultivars and 54 germplasm accessions (landrace's) from the Caribbean and North, Central, and South America. Based on 125 polymorphic random amplification polymorphic DNA (RAPD) bands, two major clusters, which generally correspond in seed size and geographic region to [be Mesoamerican and Andean gene pools, were observed among the landraces (GD = 0.726 ± 0.041). Four Fordhook cultivars and a landrace from the United States formed a separate cluster that is more distantly related to the small- (GD) = 0.561 ± 0.039) than to the large-seeded cluster (GD = 0.303 ± 0.022). The mean GD between the Andean and Mesoamerican (0.726), Mesoamerican and Fordhook (0.561), and Andean and Fordhook (0.303) clusters were all significant. The significant GD between the Andean and Mesoamerican groups supports the hypothesized existence of two major gene pools in lima bean. The RAPD marker diversity of the Mesoamerican group was the largest (0.1 10), followed by the Andean (0.097) and Ford hook (0.062) groups. The plot of the relationship between the coefficient of variation (cv) and sample size (number of bands) indicates that cvs as low as 10% for estimating CD between Andean and Mesoamerican lima bean accessions can be achieved by sampling as few as 100 bands.