Background: Rifampicin resistance (RR) is a key indicator of multidrug-resistant tuberculosis (MDR-TB) and 95% of the RR is associated with the mutation in the 81-bp rifampicin resistance determining region (RRDR) of the rpoB gene of Mycobacterium tuberculosis complex (MTBC). The Xpert MTB/RIF (Xpert) assay uses five overlapping molecular beacon probes (A-E) complementary to RRDR region that detect MTBC and mutations associated with RR. The objective of the study was to investigate the distribution and frequency of mutations detected by Xpert assay among Beijing and non-Beijing RR-TB isolates. Methods: A total of 205 randomly selected RR-TB specimens detected by Xpert assay were included in this study. A portion of specimens was further subjected to culture, MTBDRplus test and the positive culture isolates were genotyped by spoligotyping. Results: We found that the most frequent mutation occurred at probe E (S531L) binding region in both Beijing and non-Beijing isolates (61.9% and 66.9%, respectively). The Beijing family had higher mutation rates than non-Beijing (19.0% vs 12.4%) at probe B (D516V) while the non-Beijing family had higher mutations at probe D (H526D or H526Y) than the Beijing (13.2% vs 10.7%) family. Mutations at probes Aand C were less common in both Beijing and non-Beijing isolates. There was no significant difference (P=0.36) in the occurrence of mutations at different probes between Beijing and non-Beijing isolates. Conclusions: The study results revealed that the most frequent mutation occurs in the region of probe E and the least common mutations at probe A and C among both Beijing and non-Beijing RR-TB cases. This first insight into the probe mutation variation and frequencies among the RR-TB cases in Bangladesh forms the baseline information for further investigation.