Aim: We performed experiments to isolate DNA topoisomerase II gene from Pleurotus ostereatus and to discuss its application as a feasible and credible molecular maker in phylogenitic analysis. Methods and Results: A fragment of PosTopII was amplified from a P. ostreatus cDNA clone by nested polymerase chain reaction (PCR), using degenerate primers and primer walking. The full-length gene sequence was obtained with 3' and 5'-full RACE-PCR. The PosTopII cDNA is 4808 bp length and contains an open reading frame (ORF) of 4713 bp. The predicted peptide has 1571 amino acids, and exhibits strong sequence homology to other eukaryotic topoisomerase II. The PosTopII sequence was compared with the homologous genes to determine it can be used as a reliable molecular maker for P. ostreatus. Conclusions: The complete sequence of PosTopII cDNA clone was obtained. The phylogenetic relationships were consistent with other classification methods based on the morpological chracteristics and rDNA gene sequences. Significance and Impact of the Study: This is the first report on isolation of a DNA topoisomerse II from P. ostreatus. Findings from this study indicate that the DNA topoisomers II can be applied as a reliable meker for taxonomic distinction in the genus Pleurotus.