We investigated the effects of prolonged exposure to heat stress on mammary gene expression in lactating dairy cows. Eighteen mid-lactation Holstein cows were housed in tie-stalls in environmental chambers and subjected to one of the 3 treatments: 1) hyperthermia and ad libitum feed intake (HS), 2) normothermia and pair-feeding relative to HS (PF), or 3) normothermia and ad libitum feed intake (CT). Hyperthermia was modulated by exposing cows to 14 d of programmed constant heat stress (THI~80) to achieve and maintain elevated rectal temperature (~40°C). For normothermia, cows were exposed to constant thermoneutrality (THI~66) to maintain normal rectal temperature (~38.5°C). After the experimental period, all cows were placed on CT for 8 d to evaluate recovery. Mammary biopsies were obtained from randomly selected, alternating rear mammary glands on d 14 and 22 (last day of treatments and 8-d recovery post-treatment, respectively), then mRNA was isolated and sequenced. Genes were considered differentially expressed (DE) when P < 0.01 and false discovery rate ≤ 0.10. On d 14, over 75 genes were DE between HS and PF or CT, of which 23 were solely induced by HS. These genes represent upregulation of functional clusters such as superoxide metabolic process and response to reactive oxygen species, and downregulation of a cluster including extracellular space. During recovery of mammary function in HS cows, 7 DE genes were downregulated and 82 upregulated from d 14 to 22. Annotated functions of downregulated genes included superoxide formation, cell death, and others, whereas upregulated functions included clusters such as cell adhesion, collagen fibril organization, extracellular matrix organization and response to amino acid stimulus, and pathways such as protein digestion and absorption, ECM-receptor interaction, focal adhesion, PI3K-Akt signaling pathway, proteoglycans, and more. Only 16 genes were DE between PF and CT on d 14, and none on d 22. We conclude that effects of prolonged exposure to heat stress on mammary gene expression are distinct from the effects of feed restriction, in lactating dairy cows.