The bovine dry period is a dynamic non-lactating phase where the mammary gland undergoes extensive cellular turnover. Utilizing RNA sequencing, we characterized novel genes and pathways involved in this process and determined the impact of dry period heat stress. Mammary tissue was collected before and during the dry period (-3, 3, 7, 14, and 25 days relative to dry-off [day 0]) from heat-stressed (HT, n = 6) or cooled (CL, n = 6) late-gestation Holstein cows. We identified 3,315 differentially expressed genes (DEGs) between late lactation and early involution, and 880 DEGs later in the involution process. DEGs, pathways, and upstream regulators during early involution support the downregulation of functions such as anabolism and milk component synthesis, and upregulation of cell death, cytoskeleton degradation, and immune response. The impact of environmental heat stress was less significant, yet genes, pathways, and upstream regulators involved in processes such as ductal branching morphogenesis, cell death, immune function, and protection against tissue stress were identified. Our research advances understanding of the mammary gland transcriptome during the dry period, and under heat stress insult. Individual genes, pathways, and upstream regulators highlighted in this study point towards potential targets for dry period manipulation and mitigation of the negative consequences of heat stress on mammary function.