Abstract: Volvariella volvacea is a tropical mushroom with variable biological efficiency which may be stabilized through isolation of single spore cultures. Eighty seven single spore cultures (VAT-1 to VAT-87) of V. volvacea strain VV132 were isolated from three fruit bodies. The single spore cultures were characterized for their growth and enzyme producing capability followed by yield trials. Maximum growth rate was recorded in VAT- 81 and maximum biomass production was recorded in VAT-15 and VAT-82. On the basis of growth characteristics, twelve single spore cultures were selected for enzyme activity and cultivation trials along with the parent strain VV-132. Maximum endo-1,4-?- glucanase (EC 3.2.1.4) activity was recorded in VAT-82 (0.158U/h/mg). Maximum xylanase (EC 3.2.1.8) activity and exo-1,4-?-glucanase (EC 3.2.1.91) activity were recorded in VAT-15 (0.155U/h/mg and 0.083U/h/mg, respectively). The laccase (EC 1.10.3.2) activity was maximum for VAT-73 (3.66U/min/mL). In comparison to parent strain V. volvacea VV132, five single spore cultures VAT-15, VAT-26, VAT-33, VAT-73 and VAT-81 had shown higher yield whereas the number of fruit bodies was higher for single spore culture VAT-81. During the present study, these five V. volvacea single spore cultures have been identified as high yielding strains.