Background: Rifampin plays an important role in tuberculosis treatment. In recent years, the introduction of molecular testing techniques has enabled the rapid detection of rifampin resistance, leading to discrepancies with conventional methods. The World Health Organization (WHO) has analyzed mutations in the rpoB gene that induce rifampin resistance and identified certain mutations causing borderline resistance, which are often undetected using conventional tests. Consequently, the WHO has lowered the rifampin resistance criterion concentration from 1.0 to 0.5 μg/mL in 7H10 and MGIT 960. The present study aimed to evaluate the impact of this change in critical concentration on the detection of borderline rifampin resistance in Mycobacterium tuberculosis. Methods: Tuberculosis strains submitted for antituberculosis drug susceptibility testing from May 2021–2022 were analyzed. Three institutions participated; the Seoul Clinical Laboratories used the agar proportion method, whereas the Samsung Medical Center and Seoul National University Bundang Hospital utilized the MGIT 960 system to test both the original and revised concentrations. Mutations were confirmed through rpoB gene sequencing for strains showing discrepancies. Results: A total of 1,596 valid susceptibility tests were conducted during the study period.Rifampin resistance was detected in 35 cases (2.19%) at 1.0 μg/mL and in 38 cases (2.38%) at 0.5 μg/mL, whereas isoniazid resistance was observed in 158 cases (9.90%). Among the three rifampin discrepancy strains, one harbored an H445L mutation, whereas the remaining two exhibited an L452P mutation. These mutations were classified as borderline resistant. Conclusion Applying the new rifampin critical concentration resulted in a 0.19% increase in resistance rate and an 8.57% increase in detection cases. Additionally, despite testing with large number of rifampin-susceptible strains, no false resistance results were obtained.Therefore, the application of the new critical concentration is considered beneficial for the management of rifampin-resistant tuberculosis.