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ABSTRACT: Inheritance of the SLICK1 allele of the prolactin receptor gene improves thermotolerance of lactating Holstein cows under humid heat stress conditions. The aim of this study was to investigate whether pre- and postweaning Holstein heifers carrying the SLICK1 allele would show physiological responses indicative of higher tolerance to heat stress in high- and low-humidity climates. A total of 101 heifer calves of two age groups heterozygous for the SLICK1 allele and 103 wild-type half-siblings were evaluated during July 2020 in 3 dairy farms in central California and 2 in south Florida. Dry bulb temperature and relative humidity data were recorded during evaluation and used to calculate the temperature-humidity index (THI). Physiological measurements were obtained between 1600 and 1900 h in California, and 1200 and 1400 h in Florida and included rectal temperature, respiration rate, skin temperature, and sweating rate. Data were analyzed via Generalized Linear Mixed Models including the main effects of genotype, state, group, sire, farm within state, and interactions, with THI included as a covariate. The correlations between THI and dependent variables were analyzed via linear regression. The average 24-h THI was higher in Florida compared with California (90 vs. 72, respectively); the main driver of the higher THI in Florida was the high relative humidity (average 85.6% in Florida vs. 36.7% in California). In Florida, the rectal temperature of slick calves was 0.4°C lower than non-slick calves (39.5 ± 0.1 vs 39.9 ± 0.1°C); no differences were detected between slick and non-slick calves in California. Regardless of genotype, heifer calves in Florida had higher respiration rate, higher rectal and skin temperatures, and lower sweating rate than in California. This study is the first to evaluate physiological responses of calves carrying the SLICK1 allele under heat stress conditions in different climates. Our findings demonstrate that the presence of this allele is associated with lower rectal temperatures in pre- and post-weaning Holstein females. According to the physiological parameters evaluated, calves raised in Florida appeared to be under more severe heat stress; in those conditions, the SLICK1 allele was advantageous to confer thermotolerance as evidenced by lower rectal temperature in slick animals.