This study aimed to better understand environmental heat stress and physiological heat strain indicators in lactating dairy cows. Sixteen heat stress indicators were derived using microenvironmental parameters that were measured at the surrounding of cows and at usual fixed locations in the barn by using handheld and fixed subarea sensors, respectively. Twenty high-producing Holstein-Friesian dairy cows (> 30.0kg/day) from an intensive dairy farm were chosen to measure respiration rate (RR), vaginal temperature (VT), and body surface temperature of forehead (FT), eye (ET), and muzzle (MT). Our results show that microenvironments measured by the handheld sensor were slightly warmer and drier than those measured by the fixed subarea sensor; however, their derived heat stress indicators correlated equally well with physiological indicators. Interestingly, ambient temperature (Ta) had the highest correlations with physiological indicators and the best classification performance in recognizing actual heat strain state. Using segmented mixed models, the determined Ta thresholds for maximum FT, mean FT, RR, maximum ET, mean ET, VT, mean MT, and maximum MT were 24.1°C, 24.2°C, 24.4°C, 24.6°C, 24.6°C, 25.3°C, 25.4°C, and 25.4°C, respectively. Thus, we concluded that the fixed subarea sensor is a reliable tool for measuring cows' microenvironments; Ta is an appropriate heat stress indicator; FT, RR, and ET are good early heat strain indicators. The results of this study could be helpful for dairy practitioners in a similar intensive setting to detect and respond to heat strain with more appropriate indicators.