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In this study mitochondrial DNA of the indigenous honey bee populations from Serbia was analysed. Three ecotypes were comprised in the investigation: Banat (B), Syenichko-Peshterski (S) and Timok (T). The results show that all investigated samples of honey bees from Serbia belong to one mtDNA haplotype of the C phylogenetic lineage, previously designated as C2D haplotype, though with three additional polymorphic sites. For the novel mtDNA haplotype, the designation C2E is proposed. The novel C2E haplotype differs from C2D in two A-T transversions and in one insertion. Moreover, within the T honey bee ecotype, two additional polymorphic sites were observed; a deletion of a T nucleotide and a T-C transition. Our results suggest that the Carniolan honey bee populations from Serbia may represent an indigenous gene pool within A. m. carnica. The mtDNA haplotype (C2D), previously found in A. m. macedonica, was found in all samples of Serbian A. m. carnica. This suggests that the C2D haplotype may be found within both subspecies. This study represents the first effort to characterize the indigenous honey bee populations in Serbia using molecular techniques with the objective of improving our understanding of the phylogeography of Apis mellifera. [ABSTRACT FROM AUTHOR]