AbstractWe explored the diversification of the southern European Euphorbia verrucosa alliance applying molecular (amplified fragment length polymorphism fingerprinting [AFLP], sequencing of the nuclear ribosomal internal transcribed spacer), karyological (relative genome size estimations, chromosome counts) and morphometric methods. The AFLP data inferred four main phylogenetic lineages corresponding to western‐southern Balkan E. montenegrina, central Balkan E. serpentini, northern Balkan‐central European‐north Italian E. verrucosa and Iberian‐southern French E. flavicoma. Genetic diversification is strongest within the Iberian and the Balkan peninsulas, suggesting Pleistocene persistence of the species in different micro‐refugia. In contrast, weak genetic structure in E. verrucosa suggests Holocene (after last glacial maximum) expansion to central and western Europe, likely from a northern Balkan refugium. Karyological data provide evidence for tetraploidisation events in E. flavicoma and E. montenegrina, but not in E. verrucosa and E. serpentini. By integrating phylogenetic data with multivariate morphometric analyses, we propose a new taxonomic treatment for this group, mainly by recognising the Balkan endemics E. montenegrina and E. serpentini as independent species and by redefining the distributions of E. flavicoma and E. verrucosa. Our study underlines the importance of the Balkan and Iberian peninsulas as major Pleistocene refugia.