Gut microbiota plays a central role in health and nutrient digestion and would help the host for better coping with environmental perturbations. In tropical conditions or in temperate countries during Summer, elevated ambient temperatures can cause economic losses to the pig industry. During heat stress (HS), the reduction in voluntary feed intake is the main adaptation response for reducing heat production. This lower feed intake has subsequent negative effects on pig performance. The main purpose of this study was to investigate the relationships between HS and gut microbiota composition. A better understanding of the microbiota response to HS could allow the selection for animals well adapted to HS. Genetically related pigs were raised under temperate or tropical farm conditions with mean thermal humidity indexes respectively 23 and 25.5 from 11 to 23 weeks of age. In temperate conditions, pigs were submitted to a 3-week HS challenge at 30 °C. Faecal samples were collected in all pigs at 23 weeks of age in both environments (n=1,200 samples) and at 26 weeks of age in the temperate environment (n=600). Therefore, it was possible to compare microbiota from pigs raised in a temperate environment, a tropical climate, and exposed to HS. Microbiota extracted from pigs under temperate and tropical climate had different compositions whereas pigs exposed to heat challenge or raised in tropical conditions tended to share a common microbiota. HS challenge drastically modified gut microbiota and the groups before and after the challenge could be predicted in a multilevel sparse partial least square discriminant analysis with 30 OTUs and a mean classification error rate of 14%. Our experiment suggests that microbiota can be used as biomarkers of HS exposition.