Dioecious Ilex species (holly) represent a unique plant model to search for sex related molecular markers. Little is known about the genetic and physiological basis for sex determination in this genus and modern molecular marker techniques may provide tools for studying its mechanism, as well as assist in breeding and plant propagation programs to identify the female berry-producing sex at the seedling stage. In this study, female I. crenata Sky Pencil cv., male I. crenata Microphylla cv. and their F1 progeny were analyzed for the presence of AFLP markers associated with sex determination. We present here the results from a bulked-segregant analysis (BSA) using AFLP markers. Seven AFLP markers which were identified as being associated with either the male or female sex, were isolated from the gels, cloned and sequenced.