A commercially available DNA strip assay (Genotype MTBDR; Hain Lifescience, Nehren, Germany) was evaluated for its ability to detect mutations conferring resistance to rifampin (RMP) and isoniazid (INH) in clinical Mycobacterium tuberculosis complex isolates. A total of 103 multidrug-resistant (MDR; i.e., at least resistant to RMP and INH) and 40 fully susceptible strains isolated in Germany in 2001 in which resistance mutations have been previously defined by DNA sequencing and real-time PCR analysis were investigated. The Genotype MTBDR assay identified 102 of the 103 MDR strains with mutations in the rpoB gene (99%) and 91 strains (88.4%) with mutations in codon 315 of katG. All 40 susceptible strains showed a wild-type MTBDR hybridization pattern. The concordance between the MTBDR assay and the DNA sequencing results was 100%. Compared to conventional drug susceptibility testing, the sensitivity and specificity were 99 and 100% for RMP resistance and 88.4 and 100% for INH resistance, respectively. In conclusion, the MTBDR assay is a rapid and easy-to-perform test for the detection of the most common mutations found in MDR M. tuberculosis strains that can readily be included in a routine laboratory work flow.