Background <p>Delamanid (DLM) is a promising drug recommended for treatment of multidrug-/rifampicin-resistant tuberculosis (MDR/RR-TB). However, there are very little data about the emergence of drug resistance in patients with the use of DLM-containing regimens. The study aimed to monitor the dynamics of susceptibility of MTB to DLM and investigate the potential mechanism conferring decreased susceptibility.</p> Methods <p>A prospective cohort study was conducted, enrolling MDR/RR-TB patients with culture-confirmed diagnoses across 10 study sites. Serial sputum samples were collected from participants receiving DLM-containing regimens at baseline (treatment initiation), week 2, week 4, and every 4 weeks thereafter until treatment completion. The in vitro susceptibility of <i>Mycobacterium tuberculosis</i> isolates to DLM was assessed using the BACTEC MGIT 960 system.</p> Results <p>A total of 263 MDR/RR-TB patients were included from 2020 to 2024 in the present study, favorable outcomes were recorded in 187 patients (71.1%). The distribution of minimal inhibit concentration (MIC) for a bacterial population to DLM was unimodal, and most isolates tested had a MIC value of &lt; 0.015&#xa0;µg/mL. The MIC₅₀ and MIC₉₀ of MTB isolates were 0.03 and 0.06&#xa0;µg/mL, respectively. Using the 0.12&#xa0;µg/mL as a proposed epidemiological cutoff value, the resistance to DLM was found in 0.76% (2/263) of MTB isolates and no mutations were identified within loci conferring DLM resistance. Additionally, the remaining 81 (30.8%) with serial isolates were included in our analysis. 70 out of 81 patients exhibited no change in DLM MICs. In contrast, the reduced susceptibility to DLM was noted in 11 patients, defined as no less than 2-fold increase in MIC value compared with that of baseline.</p> Conclusions <p>Primary DLM resistance was rare (0.76%), and resistance emergence during treatment was infrequent, though some cases of reduced susceptibility were observed.</p>