OBJECTIVE: To develop and evaluate a rapid biochip system for the determination of multidrug-resistant tuberculosis (MDR-TB) in Mycobacterium tuberculosis isolates and clinical sputum samples. DESIGN: We developed a total solution-based system, including a biochip kit, apparatus for sample preparation, hybridisation, washing and data acquisition, and dedicated software for automated diagnosis. The biochip simultaneously identifies M. tuberculosis and detects the most commonly found mutations in the rpoB, katG and inhK genes. The system was assessed with 330 mycobacterial isolates and 129 sputum samples for rifampicin (RMP), and with 205 isolates and 105 sputum samples for isoniazid (INH), and then compared to DNA sequencing and conventional drug susceptibility testing (DST). RESULTS: The entire biochip assay took 6 h. The concordance rate between the biochip assay and the DNA sequencing results was 100%. Compared to conventional DST, the concordance rates were 91.8% for isolates and 94.6% for sputum samples for RMP resistance, and 70.2% for isolates and 78.1% for sputum samples for INH resistance. CONCLUSION: The biochip system provides a simple, rapid, reliable and accurate clinical assay for the parallel detection of M. tuberculosis and prevalent MDR-TB in a 6 h procedure, using either culture isolates or sputum samples for diagnosis. © 2009 The Union.