| Summary: | Local populations of <i>Apis mellifera</i> are rapidly changing by modern beekeeping through the introduction of nonnative queens, selection and migratory beekeeping. To assess the genetic diversity of contemporary managed honey bees in Serbia, we sequenced mitochondrial <i>tRNA<sup>leu</sup>-cox2</i> intergenic region of 241 worker bees from 46 apiaries at eight localities. Nine haplotypes were observed in our samples, with C2d being the most common and widespread. To evaluate genetic diversity patterns, we compared our data with 1696 sequences from the NCBI GenBank from neighbouring countries and Serbia. All 32 detected haplotypes belonged to the Southeast Europe lineage C, with two newly described haplotypes from our sample. The most frequent haplotype was C2d, followed by C2c and C1a. To distinguish <i>A. m. carnica</i> from <i>A. m. macedonica</i>, both previously reported in Serbia, PCR-RFLP analysis on the <i>COI</i> gene segment of mtDNA was used, and the result showed only the presence of <i>A.m. carnica</i> subspecies. An MDS plot constructed on pairwise <i>F<sub>ST</sub></i> values showed significant geographical stratification. Our samples are grouped together, but distant from the Serbian dataset from the GenBank. This, with the absence of <i>A. m. macedonica</i> subspecies from its historic range of distribution in southern Serbia, indicates that honey bee populations are changing rapidly due to the anthropogenic influence.
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