Honey bees are one of the most important social insects in the world. Genetic markers are powerful tools for differentiating honey bee subspecies. The purpose of the present research was the introduction of a new barcoding marker and assessment of the accuracy and discriminatory abilities of ND4, ND4L, ND6 genes and related intergenic spacers for the phylogenetic study of Iranian honey bees. Sampling was conducted throughout Iran (30 locations). ND4–ND4L–ND6 (with their ITS regions) demonstrated the most accurate analysis for the phylogenetic study of Iranian honey bees compared with ND4–ND4L (with ITS1) and ND4L–ND6 (with ITS2, ITS3, ITS4 and ITS5 regions). Additionally, the findings illustrated that all the collected honey bee samples were A. m. meda except those collected from the Kolachay area which were classified as A. m. carnica. Ten haplotypes were identified in the Iranian honey bee populations using the mitochondrial sequences of ND4–ND4L–ND6. Two main haplogroups were identified in the distribution range of the sampled areas. The samples of haplotype 1 containing the shortest sequence length (1580 bp), found in the Qazvin, Marand, Najafabad, Firuzkuh, Gonabad, Ilam, Birjand, Dehdasht, Sarpole-Zahab, Sanandaj, Borojerd, Sari, Zahedan and Shahrekurd samples, were grouped with A. m. meda KY464957. Moreover, the samples of haplotype 3 containing the longest sequence length (1588 bp) and found in the Kerman samples, was grouped with Bileh Savar and Soltaniyeh.
