In Korea, Gloydius brevicaudus, G. ussuriensis, and G. intermedius are the primary venomous species causing human envenomation. Although these species belong to the same genus, their envenomations induce distinct clinical symptoms, and the inconsistent efficacy of antivenoms underscores the need for species-specific therapeutic strategies. However, comprehensive molecular and toxicological profiling of Korean Gloydius species has remained unexplored. In this study, we quantitatively distinguished the venoms of three Gloydius species using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. The results revealed that each species has evolved distinct venom protein profiles. To further investigate their expression regulation, we identified transcripts coding the major venomous proteins, which revealed species-specific regulatory differences at the transcriptional level. To verify the functionality of the identified transcripts, recombinant expression of two SVMPs was successfully expressed and their proteolytic activities were tested. This study establishes a systematic framework for understanding the molecular diversity of Korean Gloydius venoms and identifies species-specific toxin candidates that could serve as potential leads for the development of tailored antivenoms, diagnostic tools, and therapeutic vaccines.