Objective To detect the mutations of capreomycin (CM) resistance related genes of Mycobacterium tuberculosis (M.tb), explore the relationship between genotype and phenotype, and provide guidance for the clinical use of CM. Methods Sputum specimens of tuberculosis patients with positive acid-fast staining in a hospital from 2017 to 2020 were collected, from which M. tb strains were clinically isolated. Drug resistance phenotypes of the strains were identified through drug susceptibility testing (DST). DNA was extracted and underwent whole-genome sequencing (WGS) to detect mutations of CM-resistance-related genes tlyA, rrs, rpsL, eis, Rv0194 and Rv1258C. Relationship between CM and rifampicin (RIF) phenotypic resistance was explored. Results 123 M.tb strains were isolated, out of which 23 nonsynonymous mutation sites in 6 CM resistance-related genes were detected via WGS. DST showed that 52 strains were resistant to RIF and 4 strains were resistant to both CM and RIF. Nonsynonymous mutation sites (A1401G in rrs, A128G in rpsL, C3293T and T221C in Rv0194) were detected in CM-resis-tant strains. Correlation between CM and RIF resistance was statistically significant (P < 0.05). Conclusion CM resistance of M. tb may be related to the mutations of rrs gene A1401G, rpsL gene A128G and Rv0194 gene C3293T, and there may be cross resistance between CM and RIF, which can be used as a reference in formulating anti-tuberculosis treatment plan.