Studies of the molecular basis of adaptations seek to understand the relative importance of structural changes in proteins versus gene regulation effects as determinants of phenotype. Amino acid substitutions in gene coding sequences are well documented as causes of variation in snake venom proteins, whereas the importance of gene regulation effects on venom protein abundance and composition is less well known. Here, we use a proteomics-based approach to infer the effects of gene regulation on protein expression by comparing the relative abundance of specific, known venom proteins among different individuals in each of two species of Sistrurus rattlesnakes. Variation in the presence or absence, and in the relative amounts, of proteins was high in both species across all major protein families. Based on our empirical criteria for inferring regulatory effects (presence-absence of specific proteins and/or more than threefold variation in abundance) between 51% and 83% of S. catenatus individuals and between 40% and 63% of S. miliarius individuals showed evidence for gene regulation across the four most abundant proteins (disintegrins, phospholipase A(2)'s, serine proteinases, and snake venom metalloproteases). Thus, the effects of gene regulation should be considered an important cause of variation in the composition of whole venoms at the intraspecific level. They also suggest the need for testing the adaptive hypothesis for venom plasticity in relation to prey consumed by adult snakes. Finally, the venom variability reported may have an impact in the treatment of bite victims, highlighting the necessity of using pooled venoms as a substrate for antivenom production.