A transposon mutant library was constructed from the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) KACC10331 by Tn5 transposon mutagenesis. The susceptible rice cultivar Milyang 23 was inoculated with a total of 24 540 mutants resistant to kanamycin and 67 avirulent or reduced-pathogenicity mutant strains were selected for study. Southern hybridization verified that 84 mutant strains had single-copy insertions and their single-transposon insertion sites were identified by sequencing analysis combined with thermal asymmetric interlaced (TAIL)-PCR. The single-transposon-tagged sequences of 21 mutant strains belonged to pathogenicity-related genes previously reported in Xanthomonas species, while the other 46 single-transposon-tagged sequences included diverse functional genes encoding, five cell-wall-degrading enzymes, three fimbrial and flagella assembly regulators, five regulatory proteins, 15 metabolic regulators and 18 hypothetical proteins, which were identified as novel pathogenicity genes of Xoo.