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We present a population genetic study focused on the two subspecies of the M evolutionary lineage, A. m. mellifera and A. m. iberiensis. Nuclear and mtDNA variation was analysed in 27 bee populations from the Iberian Peninsula, France and Belgium. Microsatellite data provides compelling evidence of a barrier to neutral gene flow at the Pyrenees. In addition, they suggest isolation by distance between populations of the M lineage. Mitochondrial data support the hypothesis that the Iberian Peninsula served as glacial refugia for the honeybees of western Europe. They show two paths of post-glacial re-colonization in the extremes of the Pyrenees and suggest that the western path was more significant in the post-glacial re-colonization process. Thus, we report here on three main factors for mellifera and iberiensis subspecies differentiation: the Pyrenean barrier, isolation by distance and the post-glacial re-colonization process. [ABSTRACT FROM AUTHOR]