In order to study pathogenic mechanisms of the plant pathogen Ascochyta rabiei, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. Hygromycin B resistance (hph) was superior to geneticin resistance (nptII) for selecting transformants, and the hph gene was more efficiently expressed by the Aspergillus nidulans trpC promoter than by the Cauliflower mosaic virus 35S promoter CaMV35S. Co-cultivation on solid media for 72 h was optimal for generating transformants, but increasing the ratio of bacterial cells to conidia did not affect transformation efficiency. All hygromycin B-resistant transformants carried transfer-DNA (T-DNA) as determined by polymerase chain reaction (PCR) and the T-DNA integrations appeared to be random and in single copy as detected by Southern hybridization. Transformants remained resistant to hygromycin B in the absence of selection. Variations in colony morphology were observed in the presence of hygromycin B under different culture conditions, and a variety of altered phenotypes including reduced virulence were observed among 550 transformants. Inverse PCR was more efficient than TAIL-PCR in identifying flanking genomic sequences from T-DNA borders, and the possible causes are discussed. This transformation technique and recovery of flanking DNA using inverse PCR will provide a useful tool for genetic studies of A. rabiei.