We compared venoms of two subspecies of blunt-nosed viper Macrovipera lebetina (Linnaeus, 1758) from Southeastern Anatolia and Cyprus by two-dimensional gel electrophoresis (2D-PAGE). Additionally, peptide mass fingerprinting analysis was carried out using matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry in order to achieve preliminary protein identification from M. lebetina obtusa venom from Turkey. As a result of 2D-PAGE, statistical tests revealed some significant differences that can be considered as subspecies-specific biomarker candidates between two subspecies. Using bioinformatic analyses, proteins belonging to 11 families were identified from the venom of M. l. obtusa: phospholipase A(2), metalloproteinase, serin proteinase, disintegrin, cysteine-rich secretory protein, C-type lectin, vascular endothelial growth factor, nerve growth factor, hyaluronidase, L: -amino acid oxidase, and trypsin inhibitor. Venom of M. lebetina was studied by 2D-PAGE for the first time in the literature, and also this is the first work aiming to determine regional variations of snake venoms by this method in Turkey and Cyprus. Our preliminary results show that snake venom research deserves more attention in Turkey as well as in the toxinology field in general.