Paracoccidioides brasiliensis, a fungal pathogen of humans, switches from a filamentous spore-forming mold in the soil to a pathogenic budding-yeast in the human host. Dimorphism is regulated mainly by the temperature of incubation. Representational difference analysis (RDA) was performed between yeast cells of isolate Pb01 and from isolate Pb4940, the last growing as mycelia at the host temperature. Transcripts exhibiting increased expression during development of the yeast parasitic phase comprised those involved mainly in response to stress, transcriptional regulation and nitrogen metabolism. In this way, the isolate Pb01 increased the expression of a variety of transcripts encoding cell rescue proteins such as the heat shock protein HSP30, alpha-trehalose-phosphate synthase and DDR48 stress protein, suggesting the relevance of the defense mechanism against oxidative/heat shock stress in the fungal yeast phase. Other differentially expressed genes between the two isolates included those coding for cell wall/membrane-related proteins, suggesting the relevance of the fungal surface and it's remodeling to the dimorphism. We provide a set of novel yeast preferentially expressed genes and demonstrate the effectiveness of RDA for studying P. brasiliensis dimorphism.