Increase the efficiency of enzymatic hydrolysis of lignocellulosic biomass is a challenge to make better use of this source for biofuel and chemical production. The use of microorganisms capable of producing high concentration of enzymes for hydrolysis in low cost substrates is presented as a solution for the process. Embrapa Agroenergia is carrying out the genetic improvement of the Trichoderma harzianum CFAM-422, aiming to achieve higher levels of (hemi)cellulolytic enzyme production. As a part of this project, this work performed the enzymatic and molecular characterization of eleven T. harzianum CFAM-422 transformants previously obtained (SHIMIZU, et al., 2015), aiming at the identification of genes involved in the production of lignocellulolytic enzymes. Transformants presented different enzymatic profile when compared to the parental strain. The T-DNA insertion region was amplified by TAIL-PCR with the specific primers LB1, LB2 and LB3 and degenerate primers AD1, AD2 and AD3. The cloning and sequencing results showed that the generated fragments did not flank the T-DNA, so it is necessary to perform new TAIL-PCR with other degenerate primers in order to identify the genetic changes in these strains.