The ectomycorrhizal fungus Hebeloma velutipes consists of two biological species (BSP 16 and 17). Within BSP 17 a dikaryon was found with two divergent types of the ribosomal Internal Transcribed Spacer (ITS1 and 2). The two ITS types segregated in monokaryotic progeny of that dikaryon, showing that these different ITS types represent different alleles at homologous rDNA loci in the two nuclei. RFLP analysis of a number of strains of BSP 17 showed that the polymorphism is widespread in Europe. There was no deficiency of the heterokaryotic type, demonstrating that ITS divergence in this species is not correlated with reduced intercompatibility. A strain from North America, not assigned to a biological species, showed the same polymorphism. Cladistic analysis of the two ITS sequences showed that they were not sister groups. One of the ITS types formed a monophyletic group together with the ITS type of BSP 16, the other type formed a clade with the ITS type of H. incarnatulum (BSP 18). BSP 16 and 17 showed partial intercompatibility. However, several lines of evidence suggest that the polymorphism of BSP 17 is not the result of frequent and continuing hybridisation with BSP 16. Instead, we give arguments for the hypothesis that the polymorphism evolved in allopatry and that the two types have come together relatively recently. The results of the polymorphism indicate a potential problem for molecular identification of fungal species based on ITS fingerprinting. The results also show that no generalisations are possible about the relation of speciation (the formation of BSP) and nuclear ITS divergence.