ABSTRACT. The genus Theobroma, recently reclassified in the family Malvaceae, comprises some species with high economic potential, including the cupuí, Theobroma subincanum Mart., which has not yet been domesticated, and whose genetics and population structure are mostly unknown. This study aimed to assess the population structure and genetic diversity in natural populations of T. subincanum Mart., using inter-simple sequence repeat (ISSR) markers. A total of 59 individuals were sampled in three geographically separate populations, CFA, CMN, and CPT. Nei's genetic distance was estimated to characterize populations with the use of 13 polymorphic primers. The analysis of 4999 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 12 (4): 4998-5006 (2013) Genetic diversity in Theobroma subincana Mart. molecular variance revealed that the variability between populations (51.71%) was higher than that within populations (48.29%). Among the three populations, CPT showed the highest diversity index and percentage of polymorphism. The ISSR molecular markers were efficient and presented sufficient polymorphism to estimate genetic diversity in populations of T. subincanum Mart.