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Objectives: India has the largest burden of MDR-TB worldwide, with an annual incidence of 1,30 000 patients. Multidrug-resistant tuberculosis (MDR-TB) carries a poor prognosis, a high mortality rate, and treatment success rates as low as 65%. The mortality in India is estimated to be about 480,000 per year. The aim of the study was to evaluate Rifampicin-resistant Tuberculosis and its distribution by GeneXpert in a tertiary care hospital. Methods: A total of 2864 samples were processed from the patients attending outpatient departments and indoor wards as per the pediatrician's request. Acid-fast bacilli (AFB) smear microscopy was done on all samples by Acid-fast staining for early diagnosis followed by GeneXpert MTB/RIF (CBNAAT) testing. Specimens were transported and stored at 2–8 °C prior to processing for CBNAAT. Results were read and reported within 2 hours. Results: A total of 2864 samples were tested for TB using CBNAAT (including 645 [22.5%] extra-pulmonary and 2219 [77.5%] pulmonary samples). The test results were positive in a total of 346 (12%) samples by CBNAAT. The positivity is highest in the age group >10 years and in sputum samples (37.28%) followed by pus samples (23.26%) in pulmonary and extrapulmonary distribution. Out ZN staining was positive in 244 (8.5%) specimens. There were 102 (29.5%) CBNAAT positive specimens which showed negative results for Acid-fast bacilli (70.52% Sensitivity). Among 346 TB-positive patients, 10.46% samples were pulmonary and 17.67% were extrapulmonary. It was observed that in positive CBNAAT patients, the prevalence of Rifampicin resistance was 12.72% i.e. 44 samples which include 31(13.36%) and 13(11.40%) pulmonary and extra-pulmonary samples respectively. Among the Rifampicin resistant samples, there were 29.55% samples that were extrapulmonary. Conclusion: GeneXpert MTB/RIF is a very rapid diagnostic assay that provides information regarding the mutation pattern of RIF resistance in MTB isolates. J Microbiol Infect Dis 2021; 11(2):81-87.

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