The aim of this study was to evaluate a pyrosequencing method for the detection of Mycobacterium tuberculosis isolates resistant to rifampin and isoniazid using both clinical strains and clinical samples, comparing the results with those of the Bactec 460TB and GenoType MTBDRplus assays. In comparison to Bactec 460TB as the gold standard, the sensitivity of pyrosequencing for detecting isoniazid and rifampin resistance was 76.9% and 97.2%, respectively, for clinical strains, and the specificity was 97.2 and 97.9%, respectively. For clinical specimens, the sensitivity and specificity for both drugs were 85.7% and 100%, respectively. The overall concordance between pyrosequencing and the GenoType MTBDRplus assay for clinical strains was 99.1%, and for clinical samples, it was 98.2%. Pyrosequencing is a valuable tool for rifampin and isoniazid resistance detection.