The Australian Mycobacterium Reference Laboratory Network collected and analysed laboratory data on new cases of disease caused by Mycobacterium tuberculosis complex in the year 2001. A total of 771 cases were identified, representing an annual reporting rate of 4.0 cases of laboratory-confirmed tuberculosis per 100,000 population. The predominant specimen type was sputum, (n=369) and a further 111 were collected at bronchoscopy. Smears were positive for 214 of 369 (58.0%) sputum and 42 of 111 (37.8%) bronchoscopy specimens respectively. Seven children (male n=5, female n=2) under 10 years of age had bacteriologically confirmed tuberculosis. A total of 69 isolates (8.9%), comprising 67 M. tuberculosis, one M. africanum, and one M. bovis, were resistant to at least one of the anti-tuberculosis agents. Excluding the M. bovis isolate, 61 of 64 (93.5%) were classified as having initial resistance, three had acquired resistance, and no data were available on the presence or absence of previous treatment for four patients. Resistance to at least isoniazid and/or rifampicin was noted for 67 isolates (8.7%), with resistance to both isoniazid and rifampicin (i.e. defined as multidrug-resistant disease) observed in 12 (1.6%) isolates. All of the multidrug-resistant isolates were M. tuberculosis, 10 were from the respiratory tract. The country of birth was known for 63 of 68 (92.6%) patients with a drug-resistant strain of M. tuberculosis or M. africanum; five were Australian-born and 58 (92.1%) had migrated from a total of 22 countries. One hundred and seven respiratory specimens had a nucleic acid amplification testing performed; 89 of 90 (98.9%) smear positives were nucleic acid amplification testing positive, whilst only 13 of 17 (76.5%) smear negative specimens were nucleic acid amplification testing positive. The 2001 laboratory data reveals a stable incidence rate and level of drug resistance in isolates from Australian patients with tuberculosis.