Despite the known association of several genes to clinical Isoniazid (INH) resistance, its molecular basis remains unknown in ∼16% of clinical isolates of Mycobacterium tuberculosis (M. tb). While screening a set of Mycobacterium smegmatis (M. smegmatis) transposon mutants with altered colony morphology for differential susceptibility to INH, we found six resistant mutants and mapped their transposon insertion sites. The disrupted genes in six INH resistant mutants were homologs of M. tb ctaE, rplY, tatA, csd and tatB with one insertion mapping to the promoter region of M. smegmatis ctaE. MIC measurements indicated a wide spectrum of INH resistance in these mutants, with complementation analyses of four selected mutants with the cognate M. smegmatis genes and their M. tb homologs confirming the association of the disrupted genes with INH resistance. Our discovery of novel genes associated with INH resistance could lead to the identification of novel INH resistance mechanisms and possibly new diagnostic modalities as well.