Heat stress during the dry period (DP) impairs milk yield in the subsequent lactation. Our objective was to examine if exposure to heat stress during the DP alters milk and colostrum quality and tight junction (TJ) permeability during the subsequent lactation. Holstein cows, selected based on parity and mature equivalent milk production, were enrolled into 2 groups: heat stress (HT, access to shade, n = 12) or cooled (CL, access to shade, fans, and soakers, n = 12) during the entire DP (~46 d, temperature-humidity index ≥68). After calving, all cows were managed together and had access to shade, fans and soakers. Colostrum and milk volume and mastitis events were recorded daily until 84 d in milk (DIM, AfiFarm System). Milk samples were collected at 0, 1, 2, 7, 14, 21, 49, and 84 DIM and analyzed for somatic cell count (SCC) by flow cytometry. Blood was collected at these time points and at −7, −2, −1 d relative to calving and assayed for lactose concentrations. Mammary gland biopsies (n = 6 per treatment) were collected at 14, 42, and 84 DIM. Total RNA was extracted to analyze the expression of genes related to TJ formation (ZO1, 2, 3, and OCLN) by real-time PCR. Data were analyzed by general linear mixed models with DIM as a repeated measure. Cooled cows had greater colostrum and milk yield compared with HT cows (7.79 vs. 3.72 ± 0.79 kg; 39.93 vs. 34.27 ± 0.80 kg/d; for colostrum and milk respectively; P < 0.01). Colostrum SCC was lower for CL compared with HT cows (839 vs. 2,729 ± 518 × 1,000 cells/mL, respectively; P = 0.01), and CL cows tended to have lower milk SCC (230 vs. 441 ± 95 × 1,000 cells/mL; P = 0.11). Cooled cows had fewer cases of mastitis relative to HT cows (1 vs. 4 cases, respectively). There was no difference in blood lactose concentrations. Expression of most TJ genes in the mammary gland did not differ between CL and HT cows, however, ZO3 expression was upregulated at 14 DIM in HT cows (P = 0.04). Cows exposed to heat stress during the DP produced less colostrum and milk with higher SCC. However, blood lactose and genes related to TJ formation were not substantially impacted by DP heat stress.