Rapid detection of the second-line drug (SLD) resistant tuberculosis (TB) strains is challenging to prescribe an immediate adequate treatment and limit the transmission of SLD resistant strains. The study aimed to evaluate the performance of GenoType MTBDRsl V2.0 compared to phenotypic drug susceptibility testing (pDST:MGIT960) to detect resistance to SLD of Mycobacterium tuberculosis (MTB) isolates in Tunisia, between May 2015 and December 2019. As a matter of fact, 103 rifampicin-resistant and multidrug-resistant MTB strains were included. Discrepancies between pDST and MTBDRsl were solved by whole genome sequencing . Compared to pDST, MTBDRsl V2.0 showed a sensitivity of 92.8% (68.5%–98.7%) in detecting resistance to fluoroquinolones. As for second-line injectable drugs, it presented a sensitivity of 80.0% (49.0%–94.3%). MTBDRsl had sensitivities of 100.0% (67.5%–100.0%), 75.0% (40.9%–92.8%) and 100.0% (60.9%–100.0%) respectively for kanamycin, capreomycin and amikacin. The specificity was 100.0% for all the drugs evaluated. As for diagnosing XDR-TB, it had a sensitivity of 57.1% (25.0%–84.1%) and a specificity of 100.0% (96.1%–100.0%). MTBDRsl V2.0 showed a high performance in detecting SLD resistance with a short turnaround time compared with pDST, which made it possible to start an early treatment and to maintain a low prevalence of SLD resistance and XDR-TB in Tunisia.