Raw data.

Apiculture is a vital economic sector in Ethiopia, providing income and employment for over two million people. However, the classification of the honey bee subspecies in Ethiopia remains debatable. To shed light on this, we analysed wing geometric and classical morphometrics, mandibular gland pheromones, and COI–COII mitochondrial DNA sequences from worker honey bees collected across high, mid and low elevation gradients within Oromia, Amhara, and Southern Nations Nationalities and Peoples’ (SNNP) regions. Our results revealed significant regional morphological and pheromonal variation driven by elevation. Wing size increased with altitude, suggesting adaptive responses to elevation. Classical morphometrics supported this trend, with bees at higher elevation exhibiting larger flight structures. Regional differences in mandibular gland pheromone secretion were also observed, with workers from Amhara secreting the least quantities of these compounds, including the queen substance 9-oxo-2(E)-decenoic acid (9-ODA) and its precursor 9-hydroxy-2(E)-decenoic acid (9-HDA), as well as the worker component 10-hydroxy-2 (E)-decenoic acid (10-HDA) and its precursor 10-hydroxy-decanoic acid (10-HDAA). Furthermore, the secretion of 9-HDA and the total amount of mandibular gland pheromone significantly and negatively correlated with elevation. For mtDNA analysis, all samples from Ethiopia clustered with the Y lineage (Apis mellifera simensis) and separated from neighbouring honey bee populations of the A lineage (A. m. scutellata and A. m. monticala). Overall, our results reveal the significant influence of elevation on adaptive traits of Ethiopian honey bees, which are of the same subspecies.