For a long time, the genus Trimeresurus (sensu lateral) has been known to consist of over 40 species of Asian pit vipers. It is now divided into four recognized genera: Trimeresurus, Ovophis, Protobothrops and Tropidolaemus [1]. The arboreal Trimeresurus (sensu stricto) are indigenous to south and south-eastern Asia. It should be noted that data from morphological and mtDNA analyses suggest that Trimeresurus (sensu stricto) is possibly derived from more than one ancestral species and should be considered as a polyphyletic group [1][2][3]. However, their venom components have not been well studied except for those of the green bamboo vipers Trimeresurus stejnegeri [4]. Distinguished by a yellow-brown skin color, Trimeresurus puniceus and Trimeresurus borneensis only inhabit Sumantra, Java and adjacent areas [5]. Previous phylogenetic studies suggest that both species are primitive and closely related to several Trimeresurus species in the Indian subcontinent [1].Snake venoms are characteristic, with specific pharmacological activities and bioavailability, and thus have potential for medical applications. In addition, investigating the diversity of venom proteins may help us to understand snake systematics and their venom To explore the venom diversity of Asian pit vipers, we investigated the structure and function of venom phospholipase A 2 (PLA 2 ) derived from two primitive tree vipers Trimeresurus puniceus and Trimeresurus borneensis. We purified six novel PLA 2 s from T. puniceus venom and another three from T. borneensis venom. All cDNAs encoding these PLA 2 s except one were cloned, and the molecular masses and N-terminal sequences of the purified enzymes closely matched those predicted from the cDNA. Three contain K49 and lack a disulfide bond at C61-C91, in contrast with the D49-containing PLA 2 s in both venom species. They are less thermally stable than other K49-PLA 2 s which contain seven disulfide bonds, as indicated by a decrease of 8.8°C in the melting temperature measured by CD spectroscopy. The M110D mutation in one of the K49-PLA 2 s apparently reduced its edematous potency. A phylogenetic tree based on the aminoacid sequences of 17 K49-PLA 2 s from Asian pit viper venoms illustrates close relationships among the Trimeresurus species and intergeneric segregations. Basic D49-PLA 2 s with a unique Gly6 substitution were also purified from both venoms. They showed edema-inducing and anticoagulating activities. It is notable that acidic PLA 2 s from both venoms inhibited blood coagulation rather than platelet aggregation, and this inhibition was only partially dependent on enzyme activity. These results contribute to our understanding of the evolution of Trimeresurus pit vipers and the structure-function relationships between various subtypes of crotalid venom PLA 2 .