Simple SummaryWhen the ambient temperature exceeds the upper limit of a certain temperature range, heat stress is triggered and then negatively affects the production, reproduction, health, and welfare of livestock animals. Due to the limitations of ambient temperature alone as a representative measure of the thermal environment, heat stress is commonly assessed by thermal indices, which contain two or more environmental parameters representing the influence of heat exchanges between the animal and its environment. To understand and utilize the thermal indices better, we evaluated several thermal indices commonly used in the heat stress assessment of dairy cows. We found that the comprehensive climate index (CCI), which includes air temperature, relative humidity, wind speed, and solar radiation, showed a better relationship with the animal-based indicators (i.e., rectal temperature, skin temperature, and eye temperature) of heat stress. According to the results of this study, the CCI has the potential to replace the temperature–humidity index in quantifying the severity of heat stress in dairy cows.Many thermal indices (TIs) have been developed to quantify the severity of heat stress in dairy cows. Systematic evaluation of the representative TIs is still lacking, which may cause potential misapplication. The objectives of this study were to evaluate the theoretical and actual performance of the TIs in a temperate climate. The data were collected in freestall barns at a commercial dairy farm. The heat transfer characteristics of the TIs were examined by equivalent air temperature change (ΔTeq). One-way ANOVA and correlation were used to test the relationships between the TIs and the animal-based indicators (i.e., rectal temperature (RT), respiration rate (RR), skin temperature (ST), and eye temperature (ET)). Results showed that the warming effect of the increased relative humidity and the chilling effect of the increased wind speed was the most reflected by the equivalent temperature index (ETI) and the comprehensive climate index (CCI), respectively. Only the equivalent temperature index for cows (ETIC) reflected that warming effect of solar radiation could obviously increase with increasing Ta. The THI and ETIC showed expected relationships with the RT and RR, whereas the CCI and ETIC showed expected relationships with the ST and ET. Moreover, CCI showed a higher correlation with RT (r = 0.672, p < 0.01), ST(r = 0.845, p < 0.01), and ET (r = 0.617, p < 0.01) than other TIs (p < 0.0001). ETIC showed the highest correlation with RR (r = 0.850, p < 0.01). These findings demonstrated that the CCI could be the most promising thermal index to assess heat stress for housed dairy cows. Future research is still needed to develop new TIs tp precisely assess the microclimates in cow buildings.