The new GenoType MTBDRsl assay (Hain Lifescience) detects mutations most frequently associated with resistance to fluoroquinolones (FLQ), aminoglycosides/cyclic peptides (AG/CP), and ethambutol (EMB) and thus, enables rapid identification of extensively drug-resistant (XDR) Mycobacterium tuberculosis strains. A set of 19 multidrug-resistant (MDR) strains isolated in Serbia in 2011 was tested by the MTBDRsl assay and by conventional drug susceptibility testing (DST). The sensitivity and specificity of the MTBDRsl assay were as follows: 100% for both for FLQ; 100% for both for AG/ CP; and 58.3% and 85.7%, respectively, for EMB. The sensitivity for detection of XDR strains was 100%. Mutations in the gyrA, rrs, and embB genes established in local M. tuberculosis strains resistant to the respective drugs have been reported as the most prevalent in other studies as well. We recommend the MTBDRsl assay as a screening test for the preliminary detection of XDR-TB cases in Serbia, but not as a replacement of the conventional second-line DST.