The role of honeybees is demonstrated in pollination and increasing crop production. The two factors of migration and importation of queens affect biodiversity of honeybee populations. Sampling was conducted from all provinces (31 provinces) of Iran in the spring and summer seasons of 2015. The tRNAleu gene, intergenic region and partial COX2 gene were used for studying honeybee populations. The phylogenetic trees were depicted using MrBayes 3.2 software by the Bayesian method and PAUP software by the Parsimony method. Results demonstrated that intergenic region and COX2 gene could segregate subspecies and evolutionary lineages from each other. A comparison of A. m. meda with some commercial subspecies demonstrated that there were 16 nucleotide differences in the intergenic regions and partial COX2 gene; 10 and 6 nucleotide differences were found in the intergenic regions and partial COX2, respectively. Results indicated that all intergenic regions of collected samples from Iran (subspecies of A. m. meda and A. m. carnica) only had the Q sequence. This study illustrated that A. m. meda was grouped into five haplotypes. The findings demonstrated that the least nucleotide diversity and segregating sites were related to the A. m. meda subspecies (π=0.0020, S=2). Moreover, the most nucleotide diversity and segregating sites were found in subspecies of A. m. iberiensis (π =0.0075, S=17). Findings showed that samples of Chramahal va Bakhtiari, Golestan, Eastern Azarbayejan, Tehran, Southern Khorasan, Shiraz, Qazvin, Mazandaran, Lorestan, Khozestan, Kordestan, Kermanshah and Sistan-Blochestan were grouped with subspecies of A. m. meda KY464957 (honeybee worker of A. m. meda from the Ruttner Bee Collection at the Bee Research Institute at Oberursel, Germany). Furthermore, the Iranian samples of Kohkeloye va Boyerahmad, Ardabil, Zanjan, Kerman and Yazd were grouped with A. m. meda FJ357806 (identified haplotype in Hakkari of Turkey, near the Iraq border). Comparisons of honeybee subspecies demonstrated that A. m. jemenitica (Y lineage) collected from Ethiopia demonstrated the highest genetic distance compared to A. m. meda (0.024 to 0.027). Genetic structure and demography analysis showed well-structured and normal expanding populations for all subspecies and lineages of Apis genus. Analysis by Structure and Bayesian Analysis of Population Structure (BAPS) revealed the four main clusters of Apis genus in all lineages, concordant with the results of the phylogenetic trees and MDS analysis. These four significant clusters included A, C, Y and Z lineages of honeybee and the M lineage was classified with the C lineage.
