Clear habitat separation between the sister species Colias alfacariensis and C. hyale is shown when occurring sympatrically. Colias hyale is found more often in moist cultivated pastures while Colias alfacariensis is more abundant in dry uncultivated habitat. Out of a total of 16 loci, no diagnostic loci were found between C. alfacariensis and C. hyale, and both species shared most major polymorphisms. Exceptions were the marked differences in allele frequencies at the HK locus and only C. hyale, but not C. alfacariensis was further invariable at the GOT2 locus, which is usually highly polymorphic in the Pieridae. Colias hyale has a significantly lower level of heterozygosity than its sister species C. alfacariensis. In Colias alfacariensis heterozygosity is highest in the Alps and lowest in the low-lying region of Northern France. Both species show high levels of gene flow over a large geographic area. Within C. alfacariensis, but not in C. hyale, the FST value of the PGI locus is significantly different from zero effectively separating the species into populations with high levels of the ’b’ allele to the west and North, and low levels of the allele in the Alps and Italy. This could point to selection within the PGI locus in line with the well established pattern of selection at the PGI locus in other species of Colias. Glaciations have been an important force in shaping the evolutionary history of European biota, leading to extinction, but also allowing new species to evolve into the newly available land as the ice sheets retreated. The genetic and distributional pattern found between both Colias species suggests that habitat shifts and subsequent adaptation during glaciations could have played an important role in their speciation.