As part of research to implement methods for the identification of Ganoderma species, this paper describes the use of PCR coupled to restriction digestions, single-strand conformational polymorphism (SSCP) and direct sequencing to assay rDNA polymorphism in South American collections of Ganoderma. The aim of the present study was to determine whether morphologically defined groups could be discriminated by molecular markers and to examine ITS sequence variation. Nineteen isolates of subgenus Ganoderma representing seven morphological taxa, and thirty isolates of subgenus Elfvingia representing three taxa, were studied. ITS I and IT II regions of twenty-one isolates were sequenced and were cladistically analyzed. Seven additional sequences were downloaded from EMBL/Genbank for comparison. In general, agreement between groupings delimited by restriction patterns, SSCP of ITS I and ITS II was evident. Gene trees derived from ITS I, ITS II and from a combined data set, were obtained and compared. Agreement between molecular and morphological data was clear at the subgeneric level; however, at the specific level, this relationship was difficult to visualize.