Two acidic Asp49-PLA2s with Glu6 substitution and a neutral Lys49-PLA (designated Gst-K49) were cloned from G. strauchii venom glands, their full amino acid sequences were deduced. The predominant acidic PLA2 (designated Gst-E6a) contains 124 residues and the M18W30 substitutions, while the minor acidic PLA2 (designated Gst-E6b) contains 122 residues and the V18A30 substitutions. Their sequences are most similar to those of the respective orthologous PLA2s of G. intermedius venom. Gst-E6a and Gst-E6b appear to be paralogs and possibly have different predatory targets or functions. The LC-MS/MS results indicate the presence of only three PLA2 gene products in the crude venom, the relative expression levels were in the order of Gst-E6a ≫ Gst-E6b > Gst-K49, as confirmed by qPCR results. In contrast to other Gloydius, G. strauchii venom does not contain neurotoxic or basic anticoagulant Asp49-PLA2s, but Gst-K49 is the first Lys49-PLA2 identified in Gloydius venoms. However, its venom content is relatively low and its pI value 7.3 is much lower than those of other Lys49-PLA2s and. The Lys49-PLA2 genes appear to regress in the venom of most of Gloydius and related rattlesnake, and this evolutionary regression occurred before the dispersal of Asian pitvipers to the New World.