Isoniazid (INH) resistance was genotypically assessed in 104 (37 INH-susceptible, 67 INH-resistant) genetically unrelated Mycobacterium tuberculosis strains cultured in North Italy. The PCR products of selected regions of the katG gene, the oxyR-ahpC intergenic region, and the inhA regulatory region were analyzed utilizing the double gradient-denaturing gradient gel electrophoresis (DG-DGGE) technique and confirmed by DNA sequencing. Mutations were detected in 61 (91%) of the INH-resistant strains, the relative frequency of the mutations being 65.7% in katG, 23.9% in oxyR-ahpC, and 13.4% in inhA. Previously described alterations, invariably associated with drug resistance, accounted for 95.1% of the mutations. No alterations were found in the INH-susceptible strains. DG-DGGE analysis and DNA sequencing were equally sensitive, but the former is cheaper, easier and more robust. Rapid genotypic assessment of INH resistance by means of the methodology described here could reasonably be used in clinical mycobacteriology laboratories.