The karyotypes of nine strains including three transformants of the chestnut blight fungus Cryphonectria parasitica were analyzed by pulsed-field gel electrophoresis (PFGE) and cytology using a fluorescence microscope. Cytology of the mitotic metaphase showed n=9 for both standard strain EP155 and field strain GH2 infected by Cryphonectria hypovirus 3. Chromosomes were morphologically characterized by size, heterochromatic segment, and constriction. PFGE resolved 5 or 6 chromosomal DNA bands ranging from 3.3Mbp to 9.7Mbp, but accurate determination of the chromosome number was hampered by clumping of some bands. Banding profiles in PFGE were similar among the strains except for GH2, in which a chromosome translocation was detected by Southern blot analysis. By integrating the data from cytology and PFGE, the genome size of C. parasitica was estimated to be ca. 50Mbp. This is the first report of a cytological karyotype in the order Diaporthales.