Objectives: Tuberculosis remains one of the top three infectious disease killers. The prevalence of multidrug-resistant tuberculosis (MDR-TB) has increased substantially in the past 20 years. When drug resistance is not detected, MDR-TB patients cannot access life-saving treatment; this puts their communities at risk of ongoing MDR-TB transmission. We aimed to determine the patterns of resistance to antituberculosis drugs among Mycobacterium tuberculosis isolates from Khuzestan province in Iran. Methods: A total of 850 clinical specimens from patients suspected of active TB were cultured in 2015. Drug susceptibility testing to the first line antiTB drugs for culture positive MTB was performed on Lowenstein–Jensen medium using the proportion method. Results: Of 850 cultured specimens, 272 (32%) were culture positive for mycobacteria. Of 64 MTB isolates that were analyzed by the proportion method, 62 (96.8%) were pan-susceptible and two (3.1%) were MDR. Conclusion An important way to prevent the emergence of MDR and XDR TB, and the principles of full implementation of the strategy is directly observed treatment, short-course (DOTS). The efficient diagnosis and timely treatment of MDR-TB patients can prevent disease transmission, reduce the risk of drug resistance developing, and avoid further lung damage.