Drug resistance in tuberculosis is a significant problem in countries endemic for tuberculosis. A sensitive, specific, and high-throughput reverse line blot assay (RLBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. DNA sequencing done for 72 resistant isolates from Delhi, for baseline data, showed mutations within the rpoB core region in all RIF-resistant strains. The RLBA includes oligonucleotide probes specific for wild-type and mutant sequences, allowing sensitive detection of both genotypes in a single assay. The assay based on reverse hybridization principle simultaneously detects 13 different mutations affecting 6 independent codons, including the most prevalent mutations at positions 531 and 526. Application of the method to a panel of 292 MDR TB isolates and susceptible strains from 5 different cities in India showed 98% concordance with the sequencing results. This rapid, simple, economical, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in Mycobacterium tuberculosis.