The cultivar Ajaya (IET 8585) exhibits durable broad-spectrum resistance to bacterial blight (BB) disease of rice and is widely used as a resistance donor. The present study was carried out to decipher the genetics of BB resistance in Ajaya and map the gene(s) conferring resistance. Genetic analysis in the F2 indicated a quantitative/additive nature of resistance governed by two loci with equal effects. Linked marker analysis and allelic tests revealed that one of the resistance genes is xa5. Sequence analysis of a 244 bp region of the second exon of the gene-encoding Transcription factor IIAγ (the candidate gene for xa5) confirmed the presence of xa5. Bulked-segregant analysis (BSA) revealed the putative location of the two quantitative trait loci (QTLs)/genes associated with resistance on chromosomes 5 and 8. Composite interval mapping located the first locus on Chr. 5S exactly in the genomic region spanned by xa5 and the second locus (qtl BBR 8.1) on Chr. 8L. Owing to its differential disease reaction with a set of seven hyper-virulent isolates of Xanthomonas oryzae, a map location on Chr. 8L, which was distinct from xa13 and data from allelism tests, the second resistance locus in Ajaya was determined to be novel and was designated as xaAj. A contig map spanning xaAj was constructed in silico and the genomic region was delimited to a 13·5 kb physical interval. In silico analysis of the genomic region spanning xaAj identified four putatively expressed candidate genes, one of which could be involved in imparting BB resistance in Ajaya along with xa5.