Simple SummaryHolstein is the most popular dairy cattle breed worldwide due to its milk yield. When these cows are exposed to heat stress, they reduce feed intake and milk production in order to minimize body heat production. The large variability associated with this response appears to be genetically regulated. Therefore, we combined genomic and marker-assisted technologies with the objective to validate genetic markers associated with milk production and thermotolerance. A genome-wide association study detected six candidate single nucleotide polymorphisms (SNPs) as predictors for milk production in heat-stressed Holstein cows. Only three of these SNPs were further validated as markers for milk production and thermotolerance traits (i.e., rectal temperature and respiratory rate) in two independent Holstein cow populations. Such markers belong to genes that regulate metabolic functions needed to accomplish energy demands and minimal heat production. The results of this study revealed that heat-stressed Holstein cows with favorable markers were able to reduce rectal temperature and respiratory rate, which allowed them to maintain adequate milk production levels. In conclusion, we validated three genetic markers in heat-stressed Holstein dairy cows, which are useful to be included in selection programs to improve milk yield and tolerance to heat stress.AbstractDairy production in Holstein cows in a semiarid environment is challenging due to heat stress. Under such conditions, genetic selection for heat tolerance appears to be a useful strategy. The objective was to validate molecular markers associated with milk production and thermotolerance traits in Holstein cows managed in a hot and humid environment. Lactating cows (n= 300) exposed to a heat stress environment were genotyped using a medium-density array including 53,218 SNPs. A genome-wide association study (GWAS) detected six SNPs associated with total milk yield (MY305) that surpassed multiple testing (p< 1.14 × 10−6). These SNPs were further validated in 216 Holstein cows from two independent populations that were genotyped using the TaqMan bi-allelic discrimination method and qPCR. In these cows, only the SNPs rs8193046, rs43410971, and rs382039214, within the genesTLR4,GRM8, andSMAD3, respectively, were associated (p< 0.05) with MY305, rectal temperature (RT), and respiratory rate. Interestingly, these variables improved as the number of favorable genotypes of the SNPs increased from 0 to 3. In addition, a regression analysis detected RT as a significant predictor (R2= 0.362) for MY305 in cows with >1 favorable genotype, suggesting this close relationship was influenced by genetic markers. In conclusion, SNPs in the genesTLR4,GRM8, andSMAD3appear to be involved in the molecular mechanism that regulates milk production in cows under heat-stressed conditions. These SNPs are proposed as thermotolerance genetic markers for a selection program to improve the milk performance of lactating Holstein cows managed in a semiarid environment.