The yeast species Rhodotorula mucilaginosa is considered to be ubiquitous due to its world-wide distribution in terrestrial, freshwater and marine habitats, and to its ability to colonize a large variety of substrates. In this paper we assess the phenotypic and genetic variability of environmental isolates of R. mucilaginosa collected from natural and artificial environments in Patagonia, Argentina. A total of 97 strains were studied and sorted into three groups based on MSP-PCR fingerprinting results: A, which comprised 90% of the strains, including the type strain; and B and C which included 2 and 8% of the strains, respectively. The D1D2 sequencing did not differentiate any of the 3 groups, while ITS sequencing validated the existence of group C. This group was composed of Patagonian isolates of diverse origin. DNA-DNA reassociation studies confirmed the existence of significant genetic differences between group C and the type strain. In this study, which is the first on the intraspecific variability of a large set of R. mucilaginosa isolates, a considerable phenotypic and genetic heterogeneity was observed, however such differences were not enough to refute co-specificity. The study of Patagonian isolates allowed the detection of a genetically distinct group of R. mucilaginosa strains.