The worldwide incidence of multi-drug-resistant tuberculosis (MDR-TB) is rapidly increasing, and it has emerged as a pressing public health issue in Iran. Nevertheless, there is a scarcity of up-to-date research on the prevalence of MDR-TB in individuals with pulmonary TB in the country. In this cross-sectional study, we gathered a total of 1216 respiratory samples, each corresponding to a unique patient, from five distinct regional TB laboratories in Iran. We identified clinical isolates as Mycobacterium tuberculosis using the IS6110-based PCR assay and Xpert MTB/RIF. Drug susceptibility testing (DST) was conducted using the conventional proportion method. Out of the collected specimens, 448 tested positive for M. tuberculosis. Among these isolates, 445 (99.4%) exhibited susceptibility to the tested drugs, while 3 (0.6%) were found to be MDR. The findings from this recent study indicate that the prevalence of MDR in Iran stands at 0.6%. The absence of recently approved treatment protocols in various regions of Iran, along with inadequately equipped laboratories lacking DST capabilities, could contribute significantly to the rise in TB/MDR-TB prevalence in Iran. Therefore, the implementation of enhanced treatment management strategies and the adoption of innovative technologies are essential steps towards improving the current situation.