Economically importance genera Trichoderma is used more frequently in crop production and plant agriculture, for high yield and in disease management as biological control agents. In present study genetic characterization (rRNA gene amplification) of twenty four indigenous isolates of Trichoderma was carried out through these standard protocols. Biochemical analysis regarding different enzymes was also performed to identify the variability of isolates on the basis of their enzyme production potential. The species selected were Trichoderma koningii, T. pseudokoningii, T. hamatum and T. harzianum. The present investigation sheds light on the nature of molecular and biochemical characterizations of some Trichoderma species and regarding inter/intra specific variations. This data will be useful for identifying fungi and in sequence analysis of rRNA as a next step. The study clearly indicates that some local strains of Trichoderma species like T. harzianum and T. koningii can be exploited in industrial production of fruit juices by cellulase enzyme potential along with further genetic engineering of the selected isolates.