Mycobacterium tuberculosis ketol-acid reductoisomerases have been widely studied due to their metabolic importance towards development of drug-resistant bacteria treatment. We here report the biochemical characterization of a new KARI (MtKARI-II) from a Mycobacterium tuberculosis variant with a similar kinetic profile to class I KARIs. Phylogenetic analysis suggested that MtKARI-II is clustered into a class II KARI superfamily.