The identification of Trichoderma strains is important for their application as biocontrol agents. We used a two-dimensional analysis, in which extracellular proteins of Trichoderma harzianum strains T-35, Y and TM were first separated according to their isoelectric point and then according to their molecular mass. Chitinase activities were detected in situ after the second separation. Each of the three strains exhibited a unique pattern of 3–5 different chitinases (1 or 2 N-acetyl-β-glucosaminidases, and 2 or 4 endochitinases). These unique profiles can be used to differentiate among strains within this species. The suggested procedure makes use of a property of T. harzianum that is directly involved in the mycoparasitic interaction, making it suitable for specific biocontrol applications.