What factors influence the evolution of a heavily selected functional trait in a diverse clade? This study adopts rattlesnakes as a model group to investigate the evolutionary history of venom coagulotoxicity in the wider context of phylogenetics, natural history, and biology. Venom-induced clotting of human plasma and fibrinogen was determined and mapped onto the rattlesnake phylogenetic tree to reconstruct the evolution of coagulotoxicity across the group. Our results indicate that venom phenotype is often independent of phylogenetic relationships in rattlesnakes, suggesting the importance of diet and/or other environmental variables in driving venom evolution. Moreover, the striking inter- and intraspecific variability in venom activity on human blood highlights the considerable variability faced by physicians treating envenomation. This study is the most comprehensive effort to date to describe and characterize the evolutionary and biological aspects of coagulotoxins in rattlesnake venom. Further research at finer taxonomic levels is recommended to elucidate patterns of variation within species and lineages.