In this study,insertional mutagenesis by Agrobacterium tumefaciens mediated transformation (ATMT) was used to build a T-DNA insertion library of Colletotrichum higginsianum containing a collection of 2 000 insertion mutants. From the library,development and pathogenicitydeficient mutants were screened and T-DNA insertion copies and sites were analyzed. As a result,we isolated 2 growth-deficient mutants Ch-1-E393 and Ch-1-C135 with significant reduced growth on PDA medium,12 mutants with abnormal colonies and one pathogenicitydeficient mutant Ch-1-G090. Pathogenicity assays showed that after inoculation of the pathogenic defective mutant Ch-1-G090 in Arabidopsis,the incidence of leaf diseases was significantly reduced. Under microscope,few primary hyphae were found in the leaves and no secondary hyphae was observed in Ch-1-G090. Southern blot analysis indicated that the mutant Ch-1-G090 harbored two T-DNA insertions. Border flanking sequences of T-DNAs from these mutants were recovered by InversePCR. Sequence analyses revealed that the two T-DNA insertion sites in the mutant Ch-1-G090 were coding genes for a hypothetical protein (Ch063_10682) and an RNA processing enzyme (CH063_10671).