The primary objective of the present study is the characterization of the hybrids of monokaryon- monokaryon (monomono)crosses in mushroom breeding. We employed this technique for developing superior strains from Pleurotus species strainswith 85 mono-mono intraspecific hybrids of 7 combinations between six Pleurotus ostreatus strains and one Pleurotus florida strain.In this study, the results of analysis on hybridization rate, nuclear DNA patterns, and colors and yields of fruit-bodies, are presentedas follows. The crossability between mono-mono crossing ranges between 50 and 93.75%. The results of the analysis on thenuclear DNA patterns of 85 hybrid strains of mono-mono crosses share the nuclei of both parents, but their genetic similaritieswere predominated by either parent. The hybrid strain between P. florida and P. ostreatus showed patterns more similar to P. florida,while the hybrid strain between P. ostreatus and P. ostreatus either had patterns predominated by either parent strain. The fruitingbody colors of the mono-mono crosses mostly had combined colors of both parents but showed the tendency of being moresimilar to that of either parent. 82% of the hybrid strain indicated similar fruiting body yields compared to both parent strains,while 0% was higher and 18% were lower than both parents. The present study was able to find out and suggest superior hybridtrains by identifying the nuclear DNA patterns of hybrids between Pleurotus species as well as the characteristics of their fruitingbodies. This study expects that the advantages of the mono-mono crossing are needs to be fully utilized in mushroom breedingand it is better to develop superior strains of Pleurotus species strains together with the mono-mono crossing.