The interaction between the host and the pathogen is extremely complex and is affected by anatomical, physiological, and immunological diversity in the microenvironments, leading to phenotypic diversity of the pathogen. Phenotypic heterogeneity, defined as nongenetic variation observed in individual members of a clonal population, can have beneficial consequences especially in fluctuating stressful environmental conditions. This is all the more relevant in infections caused by Mycobacterium tuberculosis wherein the pathogen is able to survive and often establish a lifelong persistent infection in the host. Recent studies in tuberculosis patients and in animal models have documented the heterogeneous and diverging trajectories of individual lesions within a single host. Since the fate of the individual lesions appears to be determined by the local tissue environment rather than systemic response of the host, studying this heterogeneity is very relevant to ensure better control and complete eradication of the pathogen from individual lesions. The heterogeneous microenvironments greatly enhance M. tuberculosis heterogeneity influencing the growth rates, metabolic potential, stress responses, drug susceptibility, and eventual lesion resolution. Single-cell approaches such as time-lapse microscopy using microfluidic devices allow us to address cell-to-cell variations that are often lost in population-average measurements. In this review, we focus on some of the factors that could be considered as drivers of phenotypic heterogeneity in M. tuberculosis as well as highlight some of the techniques that are useful in addressing this issue.