Abstract The Meadow and Steppe viper, Vipera ursinii-renardi complex is a well-studied group that is divided into several morphological subspecies. In this study, we combine the analyses of two mitochondrial genes with 9 microsatellite markers to compare both phylogenetic signals. Whereas the signal is similar between both genomes within most subspecies, the relative relationships between subspecies are more differentiated. Moreover, the nuclear phylogenetic reconstruction supports genetic homogeneity within V. u. macrops (in contrast to mtDNA). Both genetic portions show an unexpected differentiation between a population from Bistra Mountain and other V. u. macrops populations. Globally, the microsatellite markers suggest high genetic diversity in most subspecies, even in V. u. rakosiensis which is highly threatened; only V. u. macrops showed a limited genetic diversity. Within lowland subspecies, the differentiation between populations is globally limited compared to the distance between them (except in some populations of V. u. moldavica). The limited differentiation might be the consequence of a recent isolation (few decades) of previously large populations. Nevertheless, the only way to maintain this genetic diversity and to avoid an increase in genetic differentiation between populations in the future is to recreate suitable habitats and reconnect the populations.