Many cellular processes and organismal behaviors are time-dependent, and asynchrony of these phenomena can facilitate speciation through prezygotic and postzygotic reinforcement mechanisms. The Mojave and Sonoran desert tortoises (Gopherus agassizii and G. morafkai, respectively) reside in adjoining deserts with distinct seasonal rainfall patterns and they exhibit asynchronous winter brumation and reproductive behaviors. We used whole genome sequencing of 21 individuals from the two tortoise species and an outgroup to understand genes potentially underlying these characteristics. Eighty percent (80%) of the mutations in the most diverged 1% of the genome (FST ≥ 0.63) mapped to putatively non-functional flanking regions. Diverged genes with putatively functional variation showed extensive mutations in regulatory elements, particularly in predicted promoter regions. Clusters of genes relating to UV nucleotide excision repair, mitonuclear, and homeostasis functions had mutations in these diverged regions. Genes mediating chronobiological (cell cycle, circadian, and circannual) processes were also among the most highly diverged regions (e.g., XPA and ZFHX3). Promoter mutations had significant enrichment of genes related to regulatory machinery (ARC-Mediator complex, HDACs) suggest transcriptional cascades driven by regulatory divergence may underlie the behavioral differences between these species, leading to asynchrony-based prezygotic isolation. Further investigation revealed extensive expansion of respiratory and intestinal mucins (MUC5B, MUC5AC) within Gopherus, particularly G. morafkai. This expansion could contribute to differential Mycoplasma agassizii infection rates between the two species, as mucins help clear bacterial infections. Overall, results highlight that evolution of transcription regulation, apart from protein changes, might play an important role in the divergence and reinforcement during speciation.