This study describes the karyotype of strain 270 of the yeast-like fungus Endomyces magnusii. It consists of 13 chromosomal DNA molecules, the size of which range between 1.2 and 5.7Mb producing a genome size of approximately 38Mb. By comparing the karyotype of six strains of E. magnusii, we revealed two main chromosome length polymorphisms (CLPs) associated with a pronounced difference in the total genome size (roughly 50%). Karyotype heterogeneity between two main CLPs was demonstrated by Southern analysis with three heterologous probes. The same species affiliation of six E. magnusii strains was confirmed by morphological and cytological studies, protein fingerprint comparisons, as well as restriction analysis of mitochondrial DNA and genomic Southern analysis.