A total of five protoclones were successfully cultured on PDA medium out of regenerated twenty two colonies of Termitomyces protoplast and further studied. Liquid MYG grown mycelial tissue is used for protoplast isolation by enzymatic digestionin a mixture containing Lysing enzyme 2% and Cellulase R10 2% in 0.6 M mannitol. The incubation conditions like temperature, shaking and time were standardized at 24ºC, 60 rpm and 10 hours, respectively for healthy protoplasts liberation. The purified protoplasts showed an average yield of 1.2 × 10⁷ cells/gm tissue with 31.60 ± 9.31% regeneration efficiency on specific medium and 77.12 ± 2.72% viability by FDA test. Four ISSR primers were used in this study resulting a total of 27 reproducible bands with mean value of 6.75. They showed similar banding pattern in all the lines with zero percent polymorphism ranged from 280 bp–2700 bp. The amplified rRNA-ITS gene showed ~600 bp size in gel and found a single restriction site for enzyme HaeIII in all the protoclones and parent with similar fragment size in all.