ABSTRACT The objective of this study was to determine whether infrared thermography is a useful tool for the recognition of dairy cows in a state of thermal heat stress, as well as to identify the best region of the animal to be evaluated for this recognition. Physiological variables, including rectal temperature, respiratory frequency, cardiac frequency, and panting score were recorded in 38 lactating cows. For the assessment of environmental parameters, a digital black globe thermometer (TGD-200 model) was used. Thermographic photographs of different regions of the body of cows were taken using an infrared camera (FLIR® System T300) and indicated respective superficial temperature. Physiological variables and superficial body temperature in different regions varied between genetic groups (Girolando: ½ Holstein × ½ Gir and ¾ Holstein × ¼ Gir; purebred Holstein). The environmental temperature ranged from 20.7 to 37.9 °C with a relative humidity reaching 95%. The mean rectal temperature (40.84 °C), respiratory frequency (111.36 breaths/min), and cardiac frequency (99.22 beats/min) were higher for pure Holstein than for Girolando cows. Positive correlations were found between the physiological parameters and thermographic measures. The highest positive correlation (0.74) was found between the temperature in the lateral region of the udder and rectal temperature. Thermography is a good indicator of thermal comfort. The best region to identify heat stress in cows using thermography is the lateral region of the udder.