This study investigated further the association between the Beijing family Mycobacterium tuberculosis circulating in rural China and anti-tuberculosis (TB) drug resistance. In total, 351 M. tuberculosis isolates were collected through a population-based epidemiological study, 223 (63·5%) of which were resistant to at least one anti-TB drug, including 53 (15·1%) multidrug-resistant (MDR) isolates. Spoligotyping found 243 isolates (69·2%) that belonged to the Beijing family. A major subgroup of the Beijing family identified by mycobacterial interspersed repetitive unit (MIRU) genotyping (223325173533), showed significantly higher frequencies of MDR (44·7% vs. 13·7%, OR 6·18, 95% CI 2·68–14·23), katG and rpoB mutations (31·6% vs. 9·3%, OR 4·27, 95% CI 1·86–9·80), and being clustered by IS6110 RFLP genotyping (60·5% vs. 21·0%, OR 6·14, 95% CI 2·82–13·37) in comparison with other Beijing family isolates. Our data suggest that MIRU genotype 223325173533 of the Beijing family is associated with MDR and increased transmissibility.