The clinical management of tuberculosis is a major challenge in southern Africa. The prevalence of within-host genetically heterogeneous Mycobacterium tuberculosis infection and its effect on treatment response are not well understood. We enrolled 500 patients with tuberculosis in KwaZulu-Natal and followed them through 2 months of treatment. Using mycobacterial interspersed repetitive units-variable number of tandem repeats genotyping to identify mycobacterial heterogeneity, we report the prevalence and evaluate the association of heterogeneity with treatment response. Upon initiation of treatment, 21.1% of participants harbored a heterogeneous M. tuberculosis infection; such heterogeneity was independently associated with a nearly 2-fold higher odds of persistent culture positivity after 2 months of treatment (adjusted odds ratio, 1.90; 95% confidence interval, 1.03-3.50).