Male-associated DNA sequences were analyzed in a dioecious plant, Cannabis sativa L. (family: Moraceae), which is known to have sex chromosomes. DNA was isolated from male and female plants and subjected to random amplification of polymorphic DNA. Two out of 15 primers yielded fragments of 500 and 730 bp which were detected in all male plants but not in any of the female plants tested. These two DNA fragments were cloned and used as probes in gel blot analysis of genomic DNA. When the male and female DNAs were allowed to hybridize with the 500-bp probe, no differences in patterns were observed between male and female plants. By contrast, when these DNAs were allowed to hybridize with the 730-bp probe, much more intense bands specific to male plants were detected, in addition to less intense bands that were common to both sexes. The 730-bp DNA fragment was named MADCl (male-associated DNA sequence in Cannabis sativa). The sequence of MADCl did not include a long open reading frame and it exhibited no significant similarity to previously reported sequences.