Dioscorea, commonly known as yam, is an economically, socially, and culturally-relevant tuber cropin many tropical countries. This study aimed to characterize the genetic diversity of Dioscorea alata L. and related species from Colombia (42 D. alata L., 6 D. bulbifera L., 3 D. rotundata Poir., and 3 D. trifida L. f.) through seven inter- simple sequence repeat (ISSR) markers. The plant material was collected in five departments of Colombia. The seven ISSR generated 164 bands that showed high polymorphism (83.62%). Nei-Li genetic diversity coefficient (0.66) and Shannon information index (0.2636) revealed a high level of genetic diversity. The 54 genotypes were grouped into six groups according to geographic location but not by species. We found moderate genetic differentiation (Gst = 0.13) and high gene flow (Nm = 2.7367). The analysis of molecular variance (AMOVA) showed higher variation (86%) within groups than among groups (14%). Our results suggest that the high genetic diversity in yam in Colombia can be exploited in future work on crop improvement.