The isolated and fragmented populations are highly susceptible to stochastic events, increasing the extinction risk because of the decline in putative adaptive potential and individual fitness. The population has high heterozygosity values and a moderate allelic diversity, the heterozygosity values are higher than in most other Crotalus species and snake studies. Possibly these high levels of genetic diversity can be related to a large founder size, high effective population size, multiple paternity and overlapping generations. We did not find the genetic structuring but the effective number of alleles (Ne) was 138.1. We found evidence of bottlenecks and the majority of rattlesnakes were unrelated, despite the small sample size, endemic status, the isolated and fragmented habitat. The genetic information provided in this study can be useful as a first approach to try to make informed conservation efforts for this species and also, important to preserve the habitat of this species; the endangered Abies–Pinus forest of the Nevado the Toluca Volcano.