The global variations in the gene expression pattern of drug treated (½X) and laboratory evolved drug-resistant strains (2XR and 4XR) of Mycobacterium smegmatis were obtained and compared with the M. smegmatis mc(2) 155 (WT) strain. The genes exhibiting two-fold change and p-value <0.05 under the treated conditions have been considered as differentially expressed genes (DEGs). Overall, the numbers of DEGs observed are 1529 in ½X (596-up, 933-down), 1381 (899-up, 482-down) in 2XR and 716 in 4XR (267-up, 449-down) conditions. The data is publicly available through the GEO database with accession number GSE64132.