Fluoroquinolones are the core drugs for management of multidrug-resistant tuberculosis (MDR-TB). Molecular drug susceptibility testing methods have considerable advantages for scaling up programmatic management and surveillance of drug-resistant TB. We describe misidentification of fluoroquinolone resistance by the GenoType®MTBDRsl (MTBDRsl, Hain Lifescience GmbH, Nehren, Germany) Line Probe Assay (LPA) encountered during a feasibility and validation study for the introduction of this rapid drug susceptibility test in Kinshasa, Democratic Republic of Congo. The double gyrA mutation 80Ala and 90Gly represented 57% of all fluoroquinolone mutations identified from MDR-TB patient sputum samples, as confirmed by DNA sequencing. This double mutation was previously found to be associated with susceptibility to fluoroquinolones, yet leads to absent hybridization of a wildtype band in the MTBDRsl and is thus falsely scored as resistance. Our findings suggest to interpret MTBDRsl results with caution when the interpretation is solely based on the absence of a wildtype band without confirmation by visualization of a mutant band. Performance of the MTBDRsl LPA could be improved replacing the gyrA wildtype probes by additional probes specific for well documented gyrA mutations that confer clinically relevant resistance.