This project presents a comparative proteomic analysis of crude bee venom collected from geographically and genetically distinct Apis mellifera populations from Türkiye and Cyprus. Bottom-up proteomics was performed using nanoLC–MS/MS on a high-resolution Orbitrap platform operating in data-dependent acquisition (DDA) mode. The study aimed to characterize regional differences in venom composition and to investigate potential genetic and post-translational variability in melittin, the dominant venom peptide. Proteomic profiling revealed that melittin is the predominant venom component across all populations, while regional discrimination is primarily driven by coordinated variation in moderately and low-abundance proteins, including phospholipase A2 (PLA2), apamin, secapin family peptides, and hyaluronidase. Targeted re-analysis of MS/MS spectra identified trace but reproducible O-linked glycosylation on melittin, representing the first evidence of naturally occurring O-glycosylation in this peptide. Label-free quantification was applied to assess relative protein abundances and glycoform distribution across regions.