Tree species with wide distributions often exhibit different levels of genetic structuring correlated to their environment. However, understanding how environmental heterogeneity influences genetic variation is difficult because the effects of gene flow, drift and selection are confounded. We investigated the genetic variation and its ecological correlates in a wind-pollinated Mediterranean tree species, Fraxinus angustifolia Vahl, within a recognised glacial refugium in Croatia. We sampled 11 populations from environmentally divergent habitats within the Continental and Mediterranean biogeographical regions. We combined genetic data analyses based on nuclear microsatellite loci, multivariate statistics on environmental data and ecological niche modelling (ENM). We identified a geographic structure with a high genetic diversity and low differentiation in the Continental region, which contrasted with the significantly lower genetic diversity and higher population divergence in the Mediterranean region. The positive and significant correlation between environmental and genetic distances after controlling for geographic distance suggests an important influence of ecological divergence of the sites in shaping genetic variation. The ENM provided support for niche differentiation between the populations from the Continental and Mediterranean regions, suggesting that contemporary populations may represent two divergent ecotypes. Ecotype differentiation was also supported by multivariate environmental and genetic distance analyses. Our results suggest that despite extensive gene flow in continental areas, long-term stability of heterogeneous environments have likely promoted genetic divergence of ashes in this region and can explain the present-day genetic variation patterns of these ancient populations.