The emergence of resistance to antituberculosis drugs is a relevant matter worldwide, but the retrieval of antibiograms for Mycobacterium tuberculosis is severely delayed when phenotypic methods are used. Genotypic methods allow earlier detection of resistance, although conventional approaches are cumbersome or lack sensitivity or specificity. We aimed to design a new real-time PCR method to detect rifampin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains in a single reaction tube. First, we characterized the resistant isolates in our area of Spain by DNA sequencing. Some mutation was found within the rpoB core region in all the RIF-resistant (RIF(r)) strains. Forty-six percent of the INH-resistant (INH(r)) strains showed a mutation in katG codon 315, and most of these were associated with high MICs. Eighteen of the RIF(r), INH(r), and multidrug-resistant strains sequenced were tested by our real-time PCR assay; and full concordance of the results of the PCR with the sequencing data was obtained. In addition, a blind test was performed with a panel of 15 different susceptible and resistant strains from throughout Spain, and our results were also in 100% agreement with the sequencing data. Ours is the first assay based on rapid-cycle PCR able to simultaneously detect in a single reaction tube a large variety of mutations associated with RIF resistance (12 different mutations affecting 8 independent codons, including the most prevalent mutations at positions 526 and 531) and the most frequent INH resistance mutations. Our design could be a model for new, rapid genotypic methods able to simultaneously detect a wide variety of antibiotic resistance mutations.