In order to establish the phylogenetic relationships of genus Pleurotus, internal transcribed spacer (ITS) regions of nine strains of mushrooms were amplified and sequenced. Fungi-Seq-F1 primer and fungi-Seq-R1 were perfectly matched with 2007 and 2935 kinds of fungi, respectively. These results show that these primers can be used not only in the types of mushroom used in this study, but also in DNA sequencing analysis of the ITS region of any other mushroom. A BLAST search using about 500 bp of the 5' terminus of ITS region was carried out. The observed homology between some mushrooms and the ITS region was 98-100%. In order to investigate these results, we searched the GenBank databases. At the time of our search, the ITS region of the mushrooms was unknown and could not be found in the results of our database search in GenBank. Therefore, whole DNA sequencing of ITS region of the mushroom is considered to be of critical significance in view of future phylogenetic analyses. Additionally, the sequences of four mushrooms were aligned and showed 95-98% of homology.