Allium subgenus Melanocrommyum is taxonomically one of the most complicated groups within Allium and its sectional delimitation has been changed several times. In the present study, the relationships among 136 individuals representing 28 species of the sections Acanthoprason and Asteroprason were surveyed using ISSR (inter simple sequence repeats) markers. Nine ISSR primers generated 322 polymorphic bands (on average 36 bands per primer). In the resulting neighbor joining dendrogram, most conspecific individuals were grouped into clusters, which can be regarded as evidence of the monophyly of most species. Exceptions were the positions of six single samples of A. graveolens, A. zagricum, A. shelkovnikovii, A. subakaka, and A. materculae, which were not grouped with their remaining conspecific individuals. The clusters in dendrograms were significantly correlated with morphological similarities and biogeographical distributions. The lowest molecular similarity (62%) was observed between A. shelkovnikovii and A. ubipetrense and the most similar taxa (similarity value = 93%) were A. materculae and A. graveolens (sect. Acanthoprason). The results showed that ISSR analysis is suitable to detect the relationship of closely related species. The groups constructed agree only partly with those previously published based on molecular data, and represent alternative proposals to define taxonomic groups.