In some filamentous ascomycetes, karyotypic data by conventional light microscopy are not consistent with the electrophoretic karyotypes by pulsed field gel electrophoresis (PFGE). In this study, the three methods, conventional light microscopy on asci, PFGE, and the germ tube burst method (GTBM), were compared for their concordance in karyotyping by using Nectria haematococca (anamorph: Fusarium solani). GTBM was included in the study for cytological karyotyping on mitotic metaphase in germlings of conidia. Conventional light microscopy on asci was concluded to lead to the underestimation of chromosome number in this fungus. PFGE was effective for analysing chromosomes smaller than ca 6 Mb, but did not yield a complete karyotype due to the poor resolution of larger chromosomes. GTBM was useful for determining chromosome numbers as well as chromosome morphology. Analyses by these methods revealed that each of five strains used, two homothallic and the other three heterothallic, has a unique karyotype different from others in number and size of chromosomes. The present results indicate that combination of PFGE and GTBM constitutes a powerful tool to determine karyotypes of filamentous ascomycetes including N. haematococca.