AbstractTen microsatellite loci and a partial sequence of the COII mitochondrial gene were used to investigate genetic differentiation inB. terrestris, a bumble bee of interest for its high‐value crop pollination. The analysis included eight populations from the European continent, five from Mediterranean islands (six subspecies altogether) and one from Tenerife (initially described as a colour form ofB. terrestrisbut recently considered as a separate species,B. canariensis). Eight of the 10 microsatellite loci displayed high levels of polymorphism in most populations. InB. terrestrispopulations, the total number of alleles detected per polymorphic locus ranged from 3 to 16, with observed allelic diversity from 3.8 ± 0.5 to 6.5 ± 1.4 and average calculated heterozygosities from 0.41 ± 0.09 to 0.65 ± 0.07.B. canariensisshowed a significantly lower average calculated heterozygosity (0.12 ± 0.08) and observed allelic diversity (1.5 ± 0.04) as compared to both continental and island populations ofB. terrestris. No significant differentiation was found among populations ofB. terrestrisfrom the European continent. In contrast, island populations were all significantly and most of them strongly differentiated from continental populations.B. terrestrismitochondrial DNA is characterized by a low nucleotide diversity: 0.18%± 0.07%, 0.20%± 0.04% and 0.27%± 0.04% for the continental populations, the island populations and all populations together, respectively. The only haplotype found in the Tenerife population differs by a single nucleotide substitution from the most common continental haplotype ofB. terrestris. This situation, identical to that of Tyrrhenian islands populations and quite different from that ofB. lucorum(15 substitutions betweenterrestrisandlucorummtDNA) casts doubts on the species status ofB. canariensis. The large genetic distance between the Tenerife andB. terrestrispopulations estimated from microsatellite data result, most probably, from a severe bottleneck in the Canary island population. Microsatellite and mitochondrial DNA data call for the protection of the island populations ofB. terrestrisagainst importation of bumble bees of foreign origin which are used as crop p