Mycoparasitic Trichoderma species have expanded numbers of fungal subgroup C chitinases that contain multiple carbohydrate binding modules and could thus be important for fungal cell wall degradation during the mycoparasitic attack. In this study, we analyzed the gene regulation of subgroup C chitinases in the mycoparasite Trichoderma virens. In addition to regulation by nutritional stimuli, we found complex expression patterns in different parts of the fungal colony, and also, the mode of cultivation strongly influenced subgroup C chitinase transcript levels. Thus, the regulation of these genes is governed by a combination of colony-internal and -external signals. Our results showed completely different expression profiles of subgroup C chitinase genes in T. virens than in a previous study with T. atroviride, although both fungi are potent mycoparasites. Only a few subgroup C chitinase orthologues were found in T. atroviride and T. virens, and even those showed substantially divergent gene expression patterns. Microscopic analysis revealed morphogenetic differences between T. atroviride and T. virens, which could be connected to differential subgroup C chitinase gene expression. The biological function of fungal subgroup C chitinases therefore might not be as clear-cut as previously anticipated. They could have pleiotropic roles and might be involved in both degradation of exogenous chitinous carbon sources, including other fungal cell walls, and recycling of their own cell walls during hyphal development and colony formation.