Body temperature is a vital sign that determines physical status. Infrared thermography (IRT) is more frequently used for assessing horses' temperature because of its ease of use and less contact with the horses, making it a safer measurement method. However, the accuracy of IRT remains unclear; therefore, this study aimed to assess the potential use of IRT as an alternative method for measuring horse body temperature. Temperatures were measured in 14 horses. A digital thermometer was used to collect rectal temperature (RT), whereas a thermographic camera was used for IRT at three different positions to obtain the center of body temperature (CBT), head temperature (HT), and eye temperature (ET). The protocol was performed over 30 days, repeated thrice daily: morning (6:00-8:00), afternoon (14:00-15:00), and evening (17:00-19:00). Environmental factors, including humidity, ambient temperature, wind flow, and light intensity, were recorded indirectly according to the time of day and cooling device use. Mean RT, CBT, HT, and ET were 37.33°C, 34.08°C, 35.02°C, and 35.14°C, respectively. Center of body temperature was lower than RT by an average of 3.24°C (95% confidence interval [CI], 5.4°C-1.09°C). HT was lower than RT by an average of 2.3°C (95% CI, 4.33-0.28). The eye position showed the least difference between RT and infrared temperature, with an average of 2°C (95% CI, 0.7-3.92). However, there was no significant correlation between RT and infrared temperature at any position. Spray and vaporizer use significantly affected IRT and time of day (p = 0.05). Although IRT has advantages in terms of non-invasiveness and reduced stress on horses, its accuracy and reliability may be compromised by environmental variables, which interfere with infrared measurement. Future research should specifically focus on investigating environmental factors.