Polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region of the ribosomal DNA, subsequent cutting with the endonuclease Hinf I and restriction fragment length polymorphism (RFLP) analysis have previously proved to be a good method for distinguishing the ectomycorrhizal Tylospora fibrillosa from a large number of other basidiomycetes. In the present study this method was used to screen single mycorrhizas sampled in a Norway spruce forest in South Sweden. In this forest 98% of randomly collected mycorrhizas had the macroscopical features of T. fibrillosa mycorrhizas. Fungal ITS was successfully amplified from 93% of the sampled mycorrhizas. Five major, distinct fungal RFLP patterns were found, clearly demonstrating the benefit of the molecular method. The T. fibrillosa RFLP pattern was present in 21%, type 1 in 27%, type 2 in 20%, type 3 in 11% and type 4 in 3% of the mycorrhizas. Fungal ITS of more than one origin was found in 22% of the amplifications. It can be concluded that T. fibrillosa is one of the main mycorrhizal fungi in the studied site and it is therefore likely to be of significant ecological importance. These results suggest that T. fibrillosa would be a suitable ‘model mycorrhiza’ for studies of anthropogenic influence, such as pollution, liming or forest fertilization, on population structure of spruce mycorrhizas in southern Sweden.