Abstract Mycobacterium tuberculosis resistant to effective first-line drugs (FLDs) has challenged national and global tuberculosis control programs. This study aimed to identify mutations in 4 genes related to rifampin, pyrazinamide, and ethambutol resistance among clinical isolates of M. tuberculosis from southwestern Iran. After drug susceptibility testing of 6620 M. tuberculosis clinical isolates by proportional method, a total of 24 FLD-resistant strains were included in the study. Fragments of rpoB, pncA, embB, and ubiA genes were amplified and sequenced to mine the mutations by pairwise alignment with the corresponding M. tuberculosis H37Rv genes. Phenotypic resistance to rifampin, isoniazid, and ethambutol was detected in 67, 54, and 33% (n = 16, 13, and 8) of the isolates, respectively. Of rifampin-resistant isolates, 31% (5/16) were mono-resistant, and 56% (9/16) were multidrug-resistant (MDR). In 100% of rifampin-resistant isolates, mutations were found in the rifampin resistance-determining region (RRDR) of the rpoB, with S450L substitution being the most common, especially in MDRs (77.8%, 7/9). Resistance-conferring mutations in pncA were present in 12.5% (3/24) of FLD-resistant isolates. The embB and ubiA mutations were found in 62.5 and 12.5% (5/8 and 1/8) of ethambutol-resistant isolates, respectively, of which the embB D354A was the most common substitution (37.5%, 3/8). Sixteen distinct mutations were identified, one of which was novel. The sequence analysis of the RRDR segment was the best way to detect rifampin resistance. The rpoB S450L substitution could be a helpful molecular marker to predict MDR. In other genes, no mutation was identified as a reliable marker.