Cactus pear plant (Opuntia ficus-indica (L.) Mill.) is a type of cactus with a high tolerance to heat and drought due to the geographical and climatic conditions in which it grows. The fruits and decaying cladodes of the cactus pear plant provide a suitable environment for yeasts and other microorganisms to grow. Yeast cells accumulate trehalose and glycogen as a stress metabolite under stress conditions. Our study, it was aimed to isolate stress-tolerant yeast species from cactus pear fruit and identify them with molecular methods, and determine their storage carbohydrate accumulations. Sequence analysis of the 26S rDNA-D1/D2 gene region was used for molecular identification of thermotolerant and osmotolerant yeast strains. Trehalose and glycogen accumulations of stress-tolerant yeast strains were determined enzymatically under stress conditions. All isolated strains belonging to K. marxianus, K. lactis and P. kudriavzevii yeast species were determined as thermotolerant and osmotolerant. It was determined that K. marxianus and K. lactis yeast species preferred glycogen as a stress metabolite while P. kudriavzevii yeast species accumulated trehalose. Stress tolerance and the type and amount of reserve carbohydrate were found to be various among the other isolated yeast strains of A. pullulans, H. opuntiae and P. kluyveri depending on the degree and type of stress. It has been evaluated that the isolated K. marxianus and K. lactis yeast strains have the potential to be used in the dairy industry, while the P. kudriavzevii yeast strains are suitable for use in the bioethanol and fermentation industry.