Abstract Background and Aims The distribution of cytotypes and its potential correlation with environmental variables represent a cornerstone to understanding the origin and maintenance of polyploid lineages. Although many studies have addressed this question in single species at a regional scale, only a few have attempted to decipher this enigma in groups of closely related species at a broad intercontinental geographical scale. Here, we consider ca. 20 species of a diploid-polyploid complex (Veronica subsect. Pentasepalae) of recent and rapid diversification represented in Europe and North Africa to study the frequency and distribution of cytotypes and its relationship with environmental variables. Methods A total of 680 individuals (207 populations) were sampled. Ploidy levels were determined using flow cytometry. Ecological differentiation among cytotypes was tested using climatic and environmental variables related to temperature, precipitation, vegetation and biogeographical region, among others, and by performing univariate and multivariate (constrained PCoA) analyses. Key Results Four ploidy levels (2x, 4x, 6x, 8x) were found and genome downsizing was observed to occur within the group. Plants of different ploidy level are ecologically differentiated, with hexaploids and octoploids occurring in wetter and colder habitats with a higher seasonality than diploids. A south-to-north distribution pattern was found, with diploids occupying southern refugial areas and octoploids being more frequent in northern regions of Europe above the permafrost boundary. Conclusions The distribution of cytotypes can be explained by ecological differentiation, the geographical position of refuge areas during the Quaternary climatic oscillations, as well as by ice and permafrost retreat patterns. The Balkan Peninsula constitutes the most important contact zone between cytotypes. This work provides the first comprehensive ploidy screening within V. subsect. Pentasepalae at a broad scale and indicates that polyploidy and genome downsizing might have contributed to the colonization of new habitats in a recently diverged polyploid complex.