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Pulse-field gel electrophoresis (PFGE) was used to determine the genome size and characterize karyotypic differences in isolates of the cacao biotype of Crinipellis perniciosa (C-biotype). The karyotype analysis of four isolates from Brazil revealed that this biotype could be divided into two genotypes: one presenting six chromosomal bands and the other presenting eight. The size of the chromosomes ranged from 2.7 to 5.3 Mb. The different genotypes correlate with telomere-based PCR analysis. The isolates with six chromosomal bands had two that appeared to be doublets, as shown by densitometric analysis, indicating that the haploid chromosome number for this biotype is eight. The size of the haploid genomes was estimated at approximately 30 Mb by both PFGE and Feulgen-image analysis. DNA hybridization revealed that the rDNA sequences are clustered on a single chromosome and these sequences were located on different chromosomes in an isolate dependent manner. This is the first report of genome size and chromosomal polymorphism for the C-biotype of C. perniciosa.