Chromosomal DNA molecules were obtained from eight clinical isolates of Paracoccidioides brasiliensis during the early staged of the yeast to mycelial transformation process and were separated by contour-clamped homogeneous electric field gel electrophoresis. In seven of the isolates, it was possible to separate five bands, albeit with two different migration patterns (A and B). In the remaining isolate only four bands were observed (pattern C). The isolated chromosomes showed molecular sizes ranging from 3.2 to 10 Mb according to Schizosaccharomyces pombe molecular size standards. The three different patterns observed had bands of 3.2, 3.8, 4.1, 5.2, 6.7, 7.2, 8.8, and 10 Mb. These data imply that at least 30 Mb of the genome are organized as chromosomal macromolecules. Due to the fact that P. brasiliensis spheroplasts are difficult to obtain, technical details are provided.