A new commercial strain “Mongdol” of oyster mushroom was developed by hyphal anastomosis. It was improvedwith hybridization between monokaryotic strain derived from Pleurotus ostreatus ASI 0627 and dikaryotic strain derived from P.ostreatus ASI 2929. The optimum temperature of mycelial growth and fruiting body development were 25~30oC and 12~18oC,respectively. When two different media including PDA (potato dextrose agar medium) and MCM (mushroom complete medium)were compared, the mycelial growth of this mushroom was faster in MCM than in PDA. Similar result was observed with thecontrol strain P. ostreatus ASI 2504. Analysis of the genetic characteristics of the new cultivar “Mongdol” showed a different DNAprofile as that of the control strain ASI 2504, when RAPD (Random Amplified Polymorphic DNA) primer URP3 and URP6 wereused. Fruiting body production per bottle was about 106 g using demonstration farms. The color of pileus was blackish gray andthe stipe was long. Therefore, we expect that this new strain “Mongdol” will satisfy the consumer’s demand for high qualitymushrooms.