Drug-resistant tuberculosis (TB) presents a major challenge to global TB control. To gain a better understanding of drug-resistant TB epidemiology in Malatya, Turkey, we conducted the present study using 397 Mycobacterium tuberculosis clinical isolates collected from Malatya, Turkey, in recent years (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007). Resistance to any anti-TB drug was found in 29% (114 of 397) of the study isolates, while the multidrug resistance (MDR) rate was ϳ4.5% (18 of 397). Resistances to isoniazid (15.5%) and streptomycin (13.4%) were about twice as high as resistance to rifampin (RMP) (6.3%) and ethambutol (EMB) (6.0%). Importantly, 28% (7 of 25) of the RMP-resistant isolates were non-MDR isolates, as when a significant proportion of RMPresistant isolates in a population are non-MDR, the predictive value of molecular detection of RMP resistance for MDR can be significantly reduced. Both identical and varied drug resistance patterns were seen in the same genotyping-defined clusters, suggesting that both primary and acquired resistance have contributed to the drug-resistant TB epidemic in Malatya, Turkey. In addition, drug-resistant cases were found to be more likely to be males (odds ratio [95% confidence interval], 1.82 [1.13, 2.94]), suggesting a potential role of gender in the epidemiology of drug-resistant TB in the study population. This study demonstrates that the integration of drug susceptibility testing with genotyping and epidemiological data analysis represents a useful approach to studying the epidemiology of drug-resistant TB.