B. terrestris dalmatinus is native and the most widespread Bombus terrestris subspecies which occurs at different altitudes and habitats in the Mediterranean region where commercial B. terrestris colonies have been used for pollination in greenhouses crops for more than two decades. In this study, we sampled B. terrestris from greenhouses (commercial populations, CP1-CP7), within the five km areas surrounding the greenhouses (AK, KM, DM and GB) and more distant native populations (TM, BB and FS) in the Mediterranean region to determine the genetic structure of native and commercial B. terrestris populations and to better understand genetic introgression of commercial greenhouse B. terrestris populations into the native B. t. dalmatinus populations using twenty microsatellite markers and two mitochondrial genes (COI and cyt b). Microsatellite analysis showed adequate levels of genetic diversity in both commercial and native populations. However, populations could not been clearly separated from each other (FST = 0.014-0.045; p>0.05). All populations deviated from Hardy-Weinberg equilibrium due to high inbreeding (mean Fis: 0.234). In the mitochondrial analyses, we found two haplotypes for COI and six haplotypes for cyt b gene. Genotypes belonging to the subspecies B. t. dalmatinus were determined in all populations, while genotypes belonging to the subspecies B. terrestris terrestris were determined in some commercial populations and nearby greenhouses populations. Our results thus indicate that native populations are threatened by gene flow from non native B. t. terrestris subspecies. Our data also suggest that aestivated native FS population should be primarily conserved to avoid extinction.