Variation within species can result from adaptive processes in response to abiotic or biotic variables; however, the importance of neutral processes and population structure when considering intraspecific variation are often ignored. When investigating a trait that is assumed to be under selection, it is essential to test whether variation in the trait is concordant with genetic variation resulting from neutral demographic processes. When discordance is observed between trait variation and neutral variation, selection may be occurring and variables driving variation can be investigated. Here, we compared neutral genomic and venom expression divergence in the Red Diamond Rattlesnake (Crotalus ruber) to understand if and how biotic and/or biotic factors may be driving venom evolution. We assembled a high-quality reference genome of C. ruber to test for neutral variation and estimate venom gland transcriptome expression in multiple individuals. We found discordance between neutral genomic and venom expression variation, suggesting that selection is, at least in part, contributing to trait evolution in this system. We also found significant clinal variation in venom expression for two venom gene families: Snake Venom Metalloproteases and C-type lectins; however, the remaining six venom gene families did not demonstrate significant clinal variation. This suggests that the expression of these venom families is either not variable, or their variation is driven by other factors besides neutral processes. We conclude that abiotic factors are likely not as important in explaining venom variation and that venom variation is most likely affected by biotic variables in C. ruber.