In this study, we aimed to determine the extent of genetic variation among cubios (Tropaeolum tuberosum) in a diversity micro-center in the department of Boyacá, Colombia, with morphological and ISSR (Inter Simple Sequence Repeats) molecular markers. Twenty-five cubio morphotypes were collected across different farms within the municipalities of Belén and Ventaquemada in Boyacá and were sown for subsequent morphological and molecular characterizations. Tuber features were evaluated according to descriptors of the International Potato Center, namely predominant tuber surface color, secondary tuber surface color, secondary tuber surface color distribution, predominant tuber pulp color, secondary tuber pulp color, distribution of secondary tuber pulp color, tuber shape, and depth of tuber eyes. Molecular variation was assessed via eight ISSR primers. Morphological and molecular characterizations allowed us to identify 25 cubio morphotypes with significant genetic variation in the study area. This finding is likely attributed to mechanisms maintaining genetic variability, such as naturally occurring crosses between morphotypes modulated by environmental conditions and local agricultural practices, including seed exchange and a preference for given morphotypes.