36 fungal beta-tubulin sequences were analysed to study the evolution of this gene and the phylogeny of basidiomycetes. The analysis comprises a representative selection of all major lineages of basidiomycetous fungi and some selected ascomycetes for comparison. Intron positions vary between the different lineages, but seem to be conserved in the Hymenomycetes and Ustilaginomycetes. The most conserved regions seem to be highly susceptible for introns. Splicing and branching sites of the introns are more variable in basidiomycetes than reported from other fungal groups so far. Basidiomycete monophyly was confirmed with our data in respect to the ascomycetes studied. By analysing amino acid sequences, the Hymenomycetes and the Ustilaginomycetes were resolved as monophyletic groups. The phylogeny within these two groups is similar to that obtained with other genes. Based on beta-tubulin data Naohidea sebacea, Chionosphaera apobasidialis, Jaculispora submersa, Platygloea pustulata, Platygloea disciformis and Melampsora lini, representing the Urediniomycetes, are not resolved in most analyses. The early radiation of major basidiomycetous lineages seems to be reflected in the highly conserved beta-tubulin gene.