The molecular variability has been analysed at the mitochondrial level in 288 honeybee colonies from 28 localities distributed along the four Catalonian provinces. In total sixteen different haplotypes belonging to three evolutionary lineages were found. The west European M lineage has been found at a high frequency in the four provinces and is fixed in Lérida, whereas the African A lineage shows a cline of distribution being more frequent in the northern Catalonia. The east European C lineage has been found in three colonies (one in Gerona and two from Barcelona). These results are most likely attributable to natural evolution events but the human influence should also be considered as an agent causing recent changes in the distribution of the honeybee populations. New insights about the evolution of the honeybee populations from the Iberian Peninsula are discussed in the light of the new information provided.