Significance A central question in biology is whether trait differences are the result of variation in gene number, sequence, or regulation. Snake venoms are an excellent system for addressing this question because of their genetic tractability, contributions to fitness, and high evolutionary rates. We sequenced and assembled the genome of the Tiger Rattlesnake to determine whether the simplest rattlesnake venom was the product of a simple or complex genotype. The number of venom genes greatly exceeded the number of venom proteins producing the simple phenotype, indicating regulatory mechanisms were responsible for the production of the simplest, but most toxic, rattlesnake venom. We suggest that the retention of genomic complexity may be the result of shared regulatory elements among gene-family members.