Carotenoids are natural pigments responsible for the yellow, orange, and red colors seen in various organisms. The aim of this study was to investigate the potential of yeast strains belonging to the genus Rhodotorula isolated from the Amazon Region in the production of carotenoids. Environmental samples from the Amazon Region were collected for yeast isolation. Isolates showing pigmented colonies underwent morphological and biochemical studies, as well as assessment of their potential for carotenoid production. The three best producers were identified through nucleotide sequencing of the ITS1-5.8s-ITS4 rDNA region. The top producer underwent univariate experiments to evaluate the influence of different C/N sources. Carotenoids produced were evaluated using CCD. Results showed the isolation of 13 pigmented yeasts with morphological and biochemical characteristics consistent with the genus Rhodotorula. Isolates RGM42 (601 μg/g), RTC42 (362 μg/g), and RTC45 (351 μg/g) stood out as the top carotenoid producers. These yeasts were identified as belonging to the species Rhodotorula mucilaginosa. Regarding C/N influence, R. mucilaginosa RGM42 produced the highest number of carotenoids using glucose and peptone as carbon and nitrogen sources, respectively. Isolate R. mucilaginosa RGM42 exhibited maximum growth with a peak at 72 h of bioprocess. Extracts from isolates R. mucilaginosa RGM42, R. mucilaginosa RTC42, and R. mucilaginosa RTC45 showed retention indices like β-carotene in thin-layer chromatography; however, R. mucilaginosa RGM42 presented an additional pigment in its chromatographic profile.