OBJECTIVE: To determine the prevalence of anti-tuberculosis drug resistance in Japan. DESIGN: A nationwide drug resistance survey was conducted by convenience sampling based on a voluntary hospital consortium. Mycobacterium tuberculosis isolates (n = 2292) were collected from August 2007 to July 2008. Drug susceptibilities were analysed according to the patients' treatment history, age, sex, comorbidities and residential area. We determined susceptibility to the following drugs: isoniazid (INH), rifampicin (RMP), streptomycin (SM), ethambutol (EMB) and levofloxacin (LVX). RESULTS: The frequencies of drug-resistant isolates from new cases were as follows: INH, 3.1%; RMP, 0.7%; SM, 5.6%; EMB, 1.3%; and 8.5% to any drug. The frequencies of drug-resistant isolates from previously treated patients were as follows: INH, 12.3%; RMP, 6.7%; SM, 12.3%; and EMB, 2.6%. The frequencies of LVX-resistant isolates from new and previously treated patients were respectively 3.2% and 6.1% (n = 852). The frequencies of multidrug-resistant isolates from new and previously treated patients were respectively 0.4% and 4.1%, with only one extensively drug-resistant case. CONCLUSION: In general, the prevalence of drug-resistant TB in Japan during this period was low. However, LVX resistance among new patients was relatively high. Japan should establish a nationwide surveillance system.