The Snake Venom Detection Kit (SVDK) is of major medical importance in Australia, yet it has never been rigorously characterised in terms of its sensitivity and specificity, especially when it comes to reports of false-negative and false-positive results. This study investigates reactions and cross-reactions of five venoms the SVDK is directed against and a number of purified toxins. Snakes showing the closest evolutionary relationships demonstrated the lowest level of cross-reactivity between groups. This was, instead, far more evident between snakes that are extraordinarily evolutionary separated. These snakes: Pseudechis australis, Acanthophis antarcticus and Notechis scutatus, in fact displayed more false-positive results. Examination of individual toxin groups showed that phospholipase A(2)s (PLA(2)s) tends to react strongly and display considerable cross-reactivity across groups while the three-finger toxins (3FTx) reacted poorly in all but the Acanthophis well. The hook effect was evident for all venoms, particularly Oxyuranus scutellatus. The results of this study show considerable variation in toxin detection, with implications in further development of venom detection, both in Australia and other countries.