We applied a 7 loci Variable-Number-Tandem-Repeats (VNTR-7) analysis method to identify mixed infections of Mycobacterium tuberculosis and to estimate the rate of mixed infections among pulmonary tuberculosis patients in Shanghai, China. We validated the VNTR-7 method and used it to genotype an isolate from each of the 249 from pulmonary tuberculosis patients reported from the Songjiang and Chongming districts in Shanghai during 2006. We identified 14 patients with mixed infections, and the estimated rate of mixed infections was 5.6% (14/249) (95% CI 3.1%-9.2%). Mixed infections were observed more frequently among tuberculosis patients undergoing retreatment (15.6%) than among new cases (4.1%) (p<0.05), and among tuberculosis patients whose disease was caused by non-Beijing genotype strains (12.5%) versus Beijing genotype strains (3.5%) (p<0.05). The VNTR-7 method is a highly sensitive, practical tool with relatively high discriminatory power, making it useful for studying mixed infections.