Due to the prevailing importance of the tracheal system for insect respiration, hemoglobins had been considered rare exceptions in this arthropod subphylum. Here we report the identification, cloning and expression analysis of a true hemoglobin gene in the honeybee Apis mellifera (Hymenoptera). The deduced amino acid sequence covers 171 residues (19.5 kDa) and harbors all globin-typical features, including the proximal and the distal histidines. The protein has no signal peptide for transmembrane transport and was predicted to localize in the cytoplasm. The honeybee hemoglobin gene shows an ancient structure, with introns in positions B12.2 and G7.0, while most other insect globins have divergent intron positions. In situ hybridization studies showed that hemoglobin expression in the honeybee is mainly associated with the tracheal system. We also observe hemoglobin expression in the Malpighi tubes and testis. We further demonstrated that hemoglobins occur in other insect orders (Hemiptera, Coleoptera, Lepidoptera), suggesting that such genes belong to the standard repertoire of an insect genome. Phylogenetic analyses show that globins evolved along with the accepted insect systematics, with a remarkable diversification within the Diptera. Although insect hemoglobins may be in fact involved in oxygen metabolism, it remains uncertain whether they carry out a myoglobin-like function in oxygen storage and delivery.