Dairy cows with increased rectal temperature during heat stress experience lower milk yield and fertility. Given that rectal temperature during heat stress is heritable in dairy cattle, genetic selection for regulation of body temperature should reduce effects of heat stress on production. One goal of the study was to validate the relationship between genotype and heat tolerance for SNPs previously related to resistance to heat stress. A second goal was to identify new candidate gene SNPs related to resistance to heat stress. Thermotolerance was assessed in 625 lactating Holstein cows during the hottest part of the day in summer by measuring rectal temperature (a direct measurement of body temperature regulation), respiration rate (an indirect measurement body temperature regulation) and sweating rate (the major evaporative cooling mechanism in cattle). Specific genetic markers and candidate genes responsible for genetic variation in these variables were identified. For SNPs previously related to heat tolerance in genome-wide analysis, a region of BTA6 was related to rectal temperature and 3 closely-located genetic markers on BTA24 and another on BTA29 were associated with sweating rate. New candidate gene SNPs were identified for rectal temperature (n = 7), respiration rate (n = 9), and sweating rate (n = 6). The largest effect on rectal temperature was for PGR, which explained 2.1% of the phenotypic variation after adjustment for dry bulb temperature. This SNP could affect heat loss via cutaneous cooling because progesterone regulates vasodilation in the skin during local heating. ACAT2 (involved in lipid metabolism) explained the largest variation in respiration rate (3.3%) and SERPINE2, which regulates the enzyme thrombin that regulates epithelial cells in sweat glands, explained the largest variation in sweating rate (2.1%). These genetic markers could prove useful in genetic selection for heat tolerance in Holstein cattle.