The genetic characterization of the Iranian honey bee was investigated by analyzing 10 polymorphic DNA microsatellite loci in 300 honey bee samples representative of twenty Iranian provinces. This study evaluated the heterozygosity (Ho and He), the Shannon index, the number of observed alleles, and F-statistics among tested populations as genetic parameters. Our finding demonstrated that the Iranian honey bee populations were described by low genetic diversity in terms of the number of observed alleles, Shannon index, and Heterozygosity values. Most populations had significant deviations from Hardy Weinberg equilibrium cause of heterozygote shortage. Low F and F values proposed the absence or very low genetic diversity within and among A. m. meda populations in the present study. The cluster analysis has categorized the honey bee samples gathered from various regions of Iran into two main groups, including honey bees in the North-West (i.e., North, Northwest, and West) provinces and honey bees in the East-South (i.e., Eastern North, Central part, and Southern) provinces of Iran. Our results also revealed lower genetic differentiation and heterozygosity among tested honey bee populations. The results from this study are consistent with previous investigations in Iran, alarming the loss of genetic diversity in the Iranian honey bee populations, which leads to more homozygosity. This study presented new data and reports on genetic structure in investigated native Iranian honey bee populations, and it will benefit future studies on selection, native biodiversity preservation and other conservation breeding projects.