Four morphological traits R1, R2, R3, and R4 of Rhizocotonia solani obtained by growing on different nitrogen sources were determined for some biochemical characteristics. A further comparative study was the impact of melanin on survival and pathogenicity of R. solani, these parameters were used to estimate the total fitness for each morphological traits. It seems that these variations were due to the development of biotypes. In the present study two isolates of R. solani were obtained: one of which is melanin rich R1 (Dark) showed the most sclerotial density and the most virulent to Phaseolus vulgaris, while the other is melanin poor R4 (Albino) showed the least sclerotial density and the least virulent. Esterase isoenzyme was used as a molecular marker to discriminate the two morphological traits, which showed highly significant biochemical variation. Two phenotypes evaluated through polyacrylamide gel electrophoresis (PAGE) for isoenzyme pattern for esterase in relation to color of fungus, pathogenicity, and its sclerotial density. Specific bands on PAGE could be associated with each of these phenotypes. In addition to presence of particular banding pattern, intensity of certain bands also helped in characterization of phenotype. Esterase is considered as an effective discriminating system in characterizing phenotypes and to detect the genetic changes involved.