Potato is the most consumed vegetable worldwide. Potato tubers contain water, starch, proteins, minerals, and vitamins. The amounts of these chemicals depend on the cultivar and growing location. When potatoes are exposed to high temperatures during the growing period, tuber yield and quality are detrimentally affected; however, there is limited knowledge about the influence of high temperatures on tuber chemical composition. With temperatures rising around the globe, the reaction of potato cultivars to high temperatures is increasingly important, and heat-induced changes, including changes in the chemical composition of tubers, should be considered. The Texas A&M University Potato Breeding Program has been selecting potato clones under high-temperature conditions for many years. Several released cultivars are considered heat-tolerant based on high marketable yields and low internal and external tuber defects. In this study, we used Raman spectroscopy (RS), an analytical tool, to determine whether heat stress causes changes in the chemical composition of tubers of ten potato cultivars. RS is a non-invasive method that requires less time and labor than conventional chemical analysis. We found drastic changes in the intensities of vibrational bands that originate from carbohydrates in the spectra acquired from tubers of heat-stressed plants compared to tubers produced by potato plants grown under normal conditions. These results demonstrate that RS could be used as a replacement or complement to conventional chemical analysis to inspect the effect of heat stress on tuber chemical composition.