Honeybees are social insects and they have an important role in pollinating plants. The molecular markers provide valuable information on different taxa of insects. Combination of molecular and morphological data can increase reliability of DNA markers. In this study, the gene sequences were obtained from the GenBank of NCBI. Performance of the mitochondrial nucleotide regions of 12S rRNA, 16S rRNA and COI genes were tested for the first time in phylogenetic studies of Apis spp. The pictures of the right forewing mounts of nine species and some of their populations were obtained from the Oberursel Honey Bee Collection and Data Bank for geometric morphometric studies. Overall, the inferred phylogenetic trees of 12S rRNA, 16S rRNA, 12S rRNA + 16S rRNA and COI using the Bayesian method were shown to be more precise in phylogenetic studies compared with the Parsimony method. 12S rRNA + 16S rRNA indicated greater capability in resolving phylogenetic relationships of Apis spp. using the Bayesian and Parsimony methods. The maximum number of Parsimony-informative sites was observed in 12S rRNA + 16S rRNA (I = 212). The geometric morphometric method provided the most similar results in species relationships with barcoding genes of 12S rRNA + 16S rRNA. Furthermore, the maximum genetic (12S rRNA + 16S rRNA) and Mahalanobis distances (geometric morphometric method) were observed between Apis mellifera Linnaeus, 1758 and Apis andreniformis F. Smith, 1858. In addition, Discriminant Function Analysis (DFA) of the residual data indicated more efficacy in differentiating the dwarf and giant species compared with Procrustes coordinates using geometric morphometric method
