Coding and non-coding RNAs (ncRNAs) are potential novel markers that can be exploited for TB diagnostics in the fight against . The current study investigated the mechanisms of transcript regulation and ncRNA signatures through Total RNA Seq and small (smRNA) RNA Seq followed by Bioinformatics analysis in Beijing and F15/LAM4/KZN (KZN) clinical strains compared to the laboratory strain. Total RNA Seq revealed differential regulation of RNA transcripts in Beijing (n = 1095) and KZN (n = 856) strains compared to the laboratory H37Rv strain. The KZN vs. H37Rv coding transcripts uniquely enriched fatty acids, steroid degradation, fructose, and mannose metabolism as well as a bacterial secretion system. In contrast, Tuberculosis and biosynthesis of siderophores KEGG pathways were enriched by the Beijing vs. H37Rv-specific transcripts. Novel sense and antisense ncRNAs, as well as the expression of these transcripts, were observed, and these targeted RNA transcripts are involved in cell wall synthesis and bacterial metabolism in a strain-specific manner. RNA transcripts identified in the current study offer insights into gene regulation of transcripts involved in the growth and metabolism of the clinically relevant KZN and Beijing strains compared to the laboratory H37Rv strain and thus can be exploited in the fight against Tuberculosis.