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Background The epidemiological characteristics of drug-resistant tuberculosis (DR-TB) is fundamental to improving the prevention and control of DR-TB. Mutations in katG315 is thought to be the most predictive molecule markers for Isoniazid (INH) resistance in Mycobacterium tuberculosis (MTB). However, mutations to these genes have not been thoroughly studied in China, and epidemiological evidence of their expression levels are especially lacking in the southwest of China, which has a high TB burden within the population. Methods MTB isolates were obtained from patients with active pulmonary tuberculosis at the TB dispensary and Chest hospital in Chongqing city between June 2003 and June 2006. Proportion methods were used to test the sensitivity to INH, RFP, SM and EMB of cultured MTB. A total of 100 MTB isolates were also randomly selected for analysis of the molecular mutation spectrum of katG by DNA sequencing. Results Totally 1089 MTB isolates that completed positive sputum cultures and evaluated for their sensitivity to the four first-line drugs among 2 777 patients with TB. The prevalence of DR-TB and multi-drug resistant tuberculosis (MDR-TB) were 27.7% (302/1 089) and 7.3% (79/1 089), respectively. The resistance to anti-TB drugs was found to be highest for SM (16·3%) and INH (14·0%). There was also a significant increase in the prevalence of resistance to RFP and EMB (P＜0.01), and an increase in MDR-TB between June 2003 and June 2004 and between July 2005 and June 2006. The total mutation rate of katG315 was 75·5% (37/49) in INH-resistant MTB, and mutation sites included S315T, S315N and S3151 with mutation rates of 81·1% (30/37), 13·5% (5/37) and 5·4% (2/37), respectively.No katG315 mutants were found in any of the 48 INH-sensitive MTB. Our preliminary diagnostic results suggest that mutations in katG315 may potentially serve as molecular markers that can be used to diagnose the resistance to anti-TB drug of INH. Conclusion In the Chongqing, DR-TB and MDR-TB are increasing, and are becoming key problems for tuberculosis control. The use of katG315 mutations as potential molecule markers for drug resistance to INH may help improve patient treatment and decrease the spread of the disease.