Aim The aim of this study was to elucidate the phylogeographical pattern of taxa composing the Vipera ursinii complex, for which the taxonomic status and the dating of splitting events have been the subject of much debate. The objectives were to delimit potential refugia and to date splitting events in order to suggest a scenario that explains the diversification of this species complex. Location Western Europe to Central Asia. Methods Sequences of the mitochondrial cytochrome b and NADH dehydrogenase subunit 4 (ND4) genes were analysed for 125 individuals from 46 locations throughout the distribution range of the complex. The phylogeographical structure was investigated using Bayesian and maximum likelihood methods. Molecular dating was performed using three calibration points to estimate the timing of diversification. Results Eighty-nine haplotypes were observed from the concatenation of the two genes. Phylogenetic inferences supported two main groups, referred to in this study as the ursinii clade and the renardi clade, within which several subclades were identified. Samples from Greece (Vipera ursinii graeca) represented the first split within the V. ursinii complex. In addition, three main periods of diversification were revealed, mainly during the Pleistocene (2.42.0 Ma, 1.4 Ma and 1.00.6 Ma). Main conclusions The present distribution of the V. ursinii complex seems to have been shaped by Quaternary climatic fluctuations, and the Balkan, Caucasus and Carpathian regions are identified in this study as probable refugia. Our results support a southnorth pattern of colonization, in contrast to the northsouth colonization previously proposed for this complex. The biogeographical history of the V. ursinii complex corroborates other biogeographical studies that have revealed an eastwest disjunction (situated near the Black Sea) within a species complex distributed throughout the Palaearctic region.