To assess the performance of the GenoType MTBDR v1, a line-probe assay (LPA), to exclude baseline resistance to fluoroquinolones (FQs) and second-line injectables (SLIs) in the Standard Treatment Regimen of Anti-tuberculosis Drugs for Patients With MDR-TB 1 (STREAM 1) trial. Direct sputum MTBDR results in the site laboratories were compared to indirect phenotypic drug susceptibility testing (pDST) results in the central laboratory, with DNA sequencing as a reference standard. Of 413 multidrug-resistant TB (MDR-TB) patients tested using MTBDR and pDST, 389 (94.2%) were FQ-susceptible and 7 (1.7%) FQ-resistant, while 17 (4.1%) had an inconclusive MTBDR result. For SLI, 372 (90.1%) were susceptible, 5 (1.2%) resistant and 36 (8.7%) inconclusive. There were 9 (2.3%) FQ discordant pDST/MTBDR results, of which 3 revealed a mutation and 5 (1.3%) SLI discordant pDST/MTBDR results, none of which were mutants on sequencing. Among the 17 FQ- and SLI MTBDR inconclusive samples, sequencing showed 1 FQ- and zero SLI-resistant results, similar to frequencies among the conclusive MTBDR . The majority of inconclusive MTBDR results were associated with low bacillary load samples (acid-fast bacilli smear-negative or scantily positive) compared to conclusive results ( < 0.001). MTBDR can facilitate the rapid exclusion of FQ and SLI resistances for enrolment in clinical trials.