Both climatic and geographic factors play an important role for the biogeographic distribution of species. The Carpathian mountain ridge has been suggested as a natural geographic divide between the two honeybee subspecies Apis mellifera carnica and A. m. macedonica. We sampled one worker from one colony each at 138 traditional apiaries located across the Carpathians spanning from the Hungarian plains to the Danube delta. All samples were sequenced at the mitochondrial tRNALeu-cox2 intergenic region and genotyped at twelve microsatellite loci. The Carpathians had only limited impact on the biogeography because both subspecies were abundant on either side of the mountain ridge. In contrast, subspecies differentiation strongly correlated with the various temperature zones in Romania. A. m. carnica is more abundant in regions with the mean average temperature below 9 °C whereas A. m. macedonica honeybees is more frequent in regions with mean temperatures above 9 °C. This range selection may have impact on the future biogeography in the light of anticipated global climatic changes.