In this study, we utilized whole genome sequencing (WGS) of clinical extremely drug-resistant tuberculosis (EDR-TB) strains collected during 2014-2020 in Beijing to detect clustered strains.A retrospective cohort study was conducted by inclusion of EDR-TB patients with positive cultures in Beijing between 2014 and 2020.A total of 95 EDR-TB patients were included in our analysis. Up on the WGS based genotyping, 94 (94/95, 98.9%) out of 95 were identified as lineage 2 (East Asia). The pairwise genomic distance analysis identified 7 clusters, ranging in size from 2 to 5 isolates. The clustering rate of EDR-TB was 21.1%; while no patients had significantly higher odds of clustering. All isolates harbor rpoB RRDR mutations that confer RIF resistance and katG or inhA promoter mutations that confer INH resistance. Of 95 EDR-TB isolates, a total of 15 mutation types were recorded in the transcriptional regulator mmpR5. In vitro susceptibility testing results revealed that 14 (14/15, 93.3%) out of 15 mutation types were resistant to CFZ; whereas only 3 (3/15, 20.0%) showed resistance to BDQ. Interestingly, 12 isolates harbored mutations within rrl locus, whereas only mutations at positions 2294 and 2296 conferred CLA resistance. Favorable outcomes of EDR-TB patients were positively associated with more effective drugs in the regimes.WGS data demonstrate limited transmission of EDR-TB in this metropolis city. WGS-based drug susceptibility predictions will bring benefits to EDR-TB patients to formulate optimal therapeutic regimens.Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.