Intersimple sequence repeat (ISSR) markers were used to evaluate the effects of in vitro culture on genetic variation in Habanero pepper (Capsicum chinense Jacq.) regeneration protocols. A total of 219 ISSR clear and reproducible fragments were generated with 13 ISSR primers in direct organogenesis, direct and indirect somatic embryos, and the embryogenic callus system. A cluster analysis was performed to express in the form of dendrogram the relationships among different regeneration systems and the genetic variability detected. Genetic distance analysis indicated that our regeneration protocols are inappropriate for micropropagation, conservation, or genetic transformation; however, they may be applicable to breeding. This is the first report on the use of molecular analysis to evaluate genetic variation of in vitro-regenerated plants of Habanero pepper using ISSR markers.