Using PEG-mediated protoplast fusion, a total of nine pfle somatic hybrids were developed between Pleurotus florida and Lentinula edodes. Only six could developed fruit bodies and the second generation was made of each through tissue culture isolate from their fruit bodies. The first generation were denoted as pfle1p, pfle1q, pfle1v, pfle 1s, pfle1o and the second generation were as pfle1pFB, pfle1qFB, pfle1vFB, pfle 1sFB, pfle1oFB. The genetic closeness, stability and variance is analyzed compared to their first parental strains using nine RAPD molecular markers. Amplified PCR bands were scored analyzed using SPSS software version 19. Polymorphisms were found calculating a total of 104 bands is 94.23% where the highest polymorphism was observed in RAPD-02, RAPD- 03, RAPD-08 and SS-11. Maximum bands (16) were generated by SRS-06 and minimum in SRS-05, ranged from 330 bp – 3000 bp and 450 bp – 2000 bp, respectively. Jaccards proximity matrix was generated from the scored data followed by dendogram including two generations with parents, using single linkage. The proximity matrix was ranged from 8.00-60.00. Two parental strains P. florida and L. edodes found to be most distantly related among all, where pfle 1pFB, pfle 1rFB were very close in genetic distance and another two hybrid lines, pfle 1vFB, pfle 1qFB were also showed the same. It was observed that the nearest neighbor showed a little variance genetically, but the hybrid lines are mostly similar with their second generation.