RAPD fingerprinting was used to study species boundaries in narrowly distributed endemic species in Antirrhinum section Sempervirentia. Based on RAPD data, similarity values within species were relatively high but pairwise similarity values among species were low. Partitioning of the overall RAPDvariation using AMOVA showed that most of the variation was found among species (58.06%), whereas the remaining phenotypic diversity was distributed among populations (25.18%) and among individuals within populations (16.76%). Comparison of the matrices of geographical distances and phenetic distances (1-Dice index) among populations using the Mantel test showed a moderate, but statistically significant correlation (r¼0.588, P < 0.01), suggesting that isolation by distance is responsible for the distribution of genetic variation among Antirrhinum populations. Phenetic relationships among Antirrhinum samples based on a Dice similarity matrix showed a clear taxonomic pattern, confirming the grouping of individuals within their own populations and the clustering of populations within species. Individuals of A. charidemi, A. valentinum and A. subbaeticum, from subsection Valentina, made up a discrete group, whereas the samples belonging to subsection Sempervirentia (A. petegasii, A. sempervirens, A. microphyllum, A. pulverulentum) clustered together. RAPD data are entirely congruent with the subsection classification scheme proposed by Fernández Casas (1997) in section Sempervirentia. However, A. subbaeticum, treated as a synonym of A. valentinum by Fernández-Casas (1997), showed an unique RAPD profile characterized by the highest number of fixed species-specific markers found in section Sempervirentia. Thus, although A. valentinum appeared the most closely related species to A. subbaeticum, molecular data suggested that this species merits taxonomic distinction.