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Mixed infection with Beijing and non-Beijing strains of Mycobacterium tuberculosis has been reported and has been suggested to mediate elevation of the reinfection rate in regions with a high incidence of tuberculosis (TB). To evaluate the prevalence of infection with both Beijing and non-Beijing strains of M. tuberculosis in eastern Taiwan, the region with the highest TB incidence in Taiwan, 185 active pulmonary TB patients were enrolled at Tzu Chi General Hospital from October 2007 to September 2008. A modified multiplex PCR method was developed to distinguish Beijing and non-Beijing strains directly using the sputum of patients. Of the 185 patients, 46.5% were infected with a Beijing strain, 42.2% were infected with a non-Beijing strain, and 11.3% were infected with both strain types. Notably, mixed infection with both strain types was not associated with TB treatment history or the high-incidence race group, aborigines. In addition, the incidence rate of mixed infection before treatment with anti-TB medication was as high as that in patients with a history of anti-TB treatment. Further analysis of antibiotic susceptibility revealed that Beijing strains alone had the highest multidrug resistance rate (17.5%), mixed infection had the highest rate of resistance to at least one drug (23.8%), and non-Beijing strains had the highest rate of sensitivity to all drugs (79.5%), implying that Beijing strains are predominant in the development of drug resistance in tuberculosis.