We searched for and compared venom phospholipase A2 (PLA2) sequences containing either Asn6 or Arg6 substitutions expressed in New World pitvipers and their Asian sister taxa within the genera Gloydius, Protobothrops, and Ovophis. Phylogenetic analyses based on 35 of these PLA2s, and multiple sequence alignments, revealed the independent evolution of three subtypes of N6-PLA2s and two distinct lineages of R6-PLA2s. The results help to pinpoint the most likely Asian sister lineages of present-day New-World pitvipers.