Rapid and accurate detection of rifampicin resistance in Mycobacterium tuberculosis is crucial for effective tuberculosis control. While molecular methods targeting the rifampicin resistance determining region (RRDR) of the rpoB gene have revolutionized resistance detection, challenges arise with mutations outside this region. This study investigates the correlation between the rpoB borderline mutations Asp435Tyr and His445Asn and phenotypic resistance in M. tuberculosis. The sequencing results revealed that 11 of the 20 isolates with the Asp435Tyr mutation also had the Asn487Ser mutation and one had the Gln429His mutation. Five of the 17 isolates with the His445Asn mutation also carried the Ile491Met mutation. The study found that the presence of additional mutations outside RRDR increased the degree of phenotypic resistance. These findings suggest that mutations outside the RRDR, that not detected widely used PCR-based tests, may play a role in the variable expression of resistance associated with the Asp435Tyr and His445Asn mutations. Understanding the impact of these mutations on rifampicin resistance is important for the development of more accurate diagnostic tests and effective treatment strategies for TB.