Abstract: The anthracnose caused by Colletotrichum gloeosporioides is a serious threat to Stylosanthes guianensis, an important tropical forage. Agrobacterium tumefaciens -mediated transformation as a tool for insertional mutagenesis, is the important way to study pathogenesis of C. gloeosporioides. Based on the previously optimized Agrobacterium -mediated genetic transformation system, a mutant library containing 4 616 putative mutants was constructed, from which some mutants were randomly selected for analysis of the copy number of T-DNA insertion, genetic stability, growth rate, spore amount, spore germination and appressorium formation. The results showed that most of the mutants had single T-DNA insertion and could be stably inherited. The growth rate, spore amount, spore germination rate, appressorium formation rate of some mutants were significantly different from the wild type.