Milk production in Colombia is mainly located at high-altitude areas, where environmental temperatures are generally low. However, in the last years, it is possible to detect high temperature combined to high relative humidity at these regions, leading animals to experience some level of heat stress. Therefore, the aim of this study was to identify the alterations in milk yield and composition of Ayrshire cows under heat stress. Data were provided by the Colombian National Union of Cattle Association and Colombian Ayrshire Producers Association. A total of 10,846 milking control records, representing 2,090 cows, from 3 different Colombian Departments (Antioquia, Boyacá and Cundinamarca), in a 2-year period, were analyzed. Milk composition was determined monthly by an automated method from a certified laboratory. The temperature-humidity index (THI) was calculated based on climatic parameters including environmental temperature and humidity, obtained from weather stations belonging to the Institute of Hydrology, Meteorology and Environmental Studies, located close to the evaluated herds. Data were analyzed using a GLM model, including month within year of evaluation, nutrition (pasture or pasture + supplementation), number of calving, days in milk, and THI as fixed effect and cow as random. For this study, THI threshold of 72 was stablished, indicating that cows were under a mild heat stress when THI > 72. During the evaluated period, environmental temperature ranged from −0.12°C to 32.69°C, the relative humidity from 0% to 73%, and THI from 58 to 83. When cows were exposed to a THI > 72 it was observed a reduction in milk yield of 0.9 kg/d (15.6 to 14.7 kg/d; P < 0.001), in milk protein yield of 21.1 g/d (495.8 to 474.7 g/d;P = 0.02) and in total solids of 71.4 g/d (1,904.3 to 1,832.9 g/d; P = 0.04) were observed. Also, there was a trend (P = 0.07) for a reduction in fat yield (561.5 to 539.2 g/d). Overall, data suggest that milk production and composition of Ayrshire cows, located in high-altitude areas of Colombia are negatively affected by a mild heat stress, which deserves further investigations to mitigate these effects.