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Phylogenetic relationships of the edible shiitake mushroom (Lentinula, Tricholomataceae) were studied using DNA sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA. The ingroup consisted of seven isolates of L. edodes from Japan and Thailand, nine isolates of L. lateritia from Borneo, Papua New Guinea, and Tasmania, and five isolates of L. novaezelandieae from New Zealand. These species designations are based on morphological species concepts in Lentinula. However, because Lentinula isolates from throughout Asia-Australasia are mating compatible, some authors treat all of these as belonging to L. edodes. The outgroup included two isolates of L. boryana from Mexico. Parsimony, distance, and maximum likelihood analyses were performed, with various combinations of taxa, characters, and character codings, and bootstrap and decay index measures of robustness. Alternate topologies were evaluated in terms of tree lengths, maximum likelihood ratios, and Templeton's nonparametric test of parsimony. Results suggest that there are four independent lineages of shiitake in Asia-Australasia, which provides partial support for the morphologically based species concepts. Lentinula novaezelandieae and L. edodes sensu stricto were supported as monophyletic, but L. lateritia appears to be paraphyletic. A corollary of this is that the morphology of L. lateritia should be plesiomorphic for shiitake, which seems plausible based on outgroup comparison. In general, there is a strong correlation between the geographic origins of the isolates and the lineages supported. Biogeographic interpretation of ITS trees suggests that the ancestral area for shiitake in Asia-Australasia is in the South Pacific, which was the most phylogenetically diverse area examined. Phylogenetic analyses including previously published ITS2 sequences of three shiitake isolates of unknown origin placed the unknown isolates in a group of L. edodes sensu stricto isolates from Japan and Thailand, which suggests that the unknown isolates are from northeast or continental Asia. ITS-based cladograms have points of agreement as well as disagreement with previously published mitochondrial DNA-based dendrograms from a subset of the isolates used in this study. Discrepancies between the ITS and mtDNA trees could mean that the nuclear ITS and the mtDNA have different evolutionary histories. However, comparison of mtDNA and ITS data are complicated by the fact that the mtDNA data are based on pairwise distances, whereas the ITS data are discrete. Practical implications of the results for shiitake breeding and conservation are discussed.