Most plant pathogens present complex life cycles, in which the clonal reproduction may impede the delimitation of population entities. By studying the genetic structure of the wheat yellow rust caused by Puccinia striiformis f.sp. tritici (PST), we highlighted difficulties impeding population delimitation in highly clonal species. Despite the high dispersal potential of PST, southern France isolates were shown to be divergent from a northwestern European population. A 2-year survey was performed in the Mediterranean region to assess the geographic distribution of southern isolates: 453 isolates collected in 11 countries were genotyped using 15 simple sequence repeat markers. A subsample was analysed for virulence against 23 resistance genes. The dominant strain in the western Mediterranean region was further studied with amplified fragment length polymorphism markers to test for a geographic substructure. Both ‘individual’- and ‘population’-centred analyses of polymorphism markers revealed two south-specific groups: a predominant group, with a broad variability and a wide distribution in both western and eastern Mediterranean countries, and a minor group in the western Mediterranean. The east–west gradient of genetic diversity suggested gene flow from the Middle East with subsequent founder effects and genetic divergence, and demonstrated the local survival of a western Mediterranean population. The high frequency of the resistance gene Yr8 observed in cultivars from Tunisia and Algeria may contribute to maintain the north/south structure observed in France. In addition to migration and local adaptation, the dynamics of clonal lineage diversification and replacement should be considered to define population entities in strongly clonal species.