AbstractThe pncA gene encodes pyrazinamidase enzyme which converts drug pyrazinamide to active form pyrazinoic acid, but mutations in this gene can prevent enzyme activity which leads to pyrazinamide resistance. The cross‐sectional study was carried out during 2016–2017 for 12 months. The purpose of the study was to detect mutation at codon 12 and codon 85 in the pncA gene in local multidrug‐resistant tuberculosis (MDR‐TB) patients by developing a simple molecular test so that disease could be detected timely in the local population. DNA extracted from sputum‐cultured samples from MDR‐TB patients and subjected to semi‐multiplex allele‐specific PCR by using self‐designed primers against the pncA gene. Among 75 samples, 53 samples were subjected to molecular analysis based on purified DNA quantity and quality. The primers produced 250 and 480 bp fragments, indicating the mutations at codon 12 (aspartate to alanine) and codon 85 (leucine to proline) respectively. MDR‐TB was more common in the age group 21–40 years. Fifty‐seven percent of samples (n = 30) were found positive for pncA mutations, whereas 43% of samples (n = 23) showed negative results. Thirteen percent of samples (n = 4) had mutations at codon 12 in which aspartate was converted to alanine, and they produced an amplified product of 480 bp. Eighty‐seven percent of samples (n = 26) had mutations at codon 85 in which leucine was converted to proline and amplified product size was 250 bp. The mutations were simple nucleotide substitutions. The prevalence of mutations in which leucine was substituted by proline was higher than the mutations in which aspartate was substituted by alanine. A high prevalence of substitution mutation (CTG → CCG; leucine to proline) was detected in MDR‐TB cases. Earlier detection of MDR‐TB via an effective molecular diagnostic method can control the MDR tuberculosis spread in the population.