Mycobacterium tuberculosis (Mtb) exhibits remarkable phenotypic diversity, enabling its adaptation to various host environments. The diversity of in vivo niches at granuloma, cellular and sub-cellular levels could promote phenotypic heterogeneity in Mtb resulting in Mtb sub-populations with differential access or response to drugs, leading to drug-tolerant sub-populations. We employ a panel of fluorescent Mtb sensor strains in mouse models to quantify phenotypic diversity across various host niches at single-bacterial resolution, allowing us to generate niche-specific phenotypic signatures for individual Mtb and identify changes upon drug treatment. Our findings reveal that Mtb exhibits significant phenotypic heterogeneity in response to different host environments. Targeting Mtb subpopulations with specific phenotypic states could offer a promising approach to overcome drug resistance and improve treatment outcomes.