Yeasts of the genus Rhodotorula have pink, orange, and red colors, indicating their ability to synthesize carotenoids. Due to their biological properties, carotenoids are widely used in the phytomedicine, chemical, pharmaceutical, cosmetic, food, and feed industries. One of the most important carotenoids is β-carotene, which exhibits interesting biological activities, such as antioxidant, anticancer, and antimicrobial. Rhodotorula yeasts were isolated from the natural environment and analyzed using morphological and molecular methods. Species were identified based on the restriction fragment length polymorphism analysis and sequencing of the rDNA internal transcribed spacer region (ITS). In this work, the β-carotene content of Rhodotorula yeasts was investigated when grown on a solid YEPD medium at different temperatures (4 °C and 26 °C) using various light intensities (8-10 µmol/m²s). The biomass was collected, and carotenoids were extracted using dimethylformamide (DMF). The amount of β-carotene was calculated based on absorption at 440 nm. The highest β-carotene contents were found in R. babjevae, R. glutinis, R. graminis, and R. kratovchilovae after 14 days of incubation at 26 °C in the dark. The level of β-carotene was 30 to 55% lower depending on the yeast species when the yeast culture after 2 days of cultivation at 26 °C was transferred to 4 °C, followed by 12 days of incubation in the dark. When yeasts were kept in the light at 4 °C, out of the eight yeast species tested, R. mucilaginosa and R. babjevae accumulated about 20-30% more β-carotene than under dark conditions. Our results show that yeasts that are cultured in the dark at 26 °C accumulate β-carotene better than yeasts that are held under the same conditions in the light. The synthesis and accumulation of β-carotene are decreased at a low temperature. The obtained data suggest that isolated Rhodotorula yeast species can be promising for carotenoid production under appropriate conditions.