Standard methods for enumerating Mycobacterium tuberculosis in patient sputum can miss large populations of viable M. tuberculosis cells that are unable to grow either on solid medium or in liquid medium unless the medium has been extensively diluted. Because these bacteria can be detected in liquid medium after limiting dilution, they have been termed differentially culturable or differentially detectable M. tuberculosis (DD-). Treatment with isoniazid (H), rifampin (R), pyrazinamide (Z), and ethambutol (E) (HRZE) for 1 to 2 weeks has been shown to increase the representation of DD- in the sputum of drug-sensitive (DS) tuberculosis (TB) patients. However, little is known about DD- after longer periods of treatment with HRZE or in patients with drug-resistant (DR) TB who receive second-line therapies. Here, we measured the proportion of DD- cells in the sputum of 47 subjects, 29 with DS TB and 18 with DR TB, before initiation of treatment and at 2 weeks and 2 months thereafter. Prior to treatment, DD- cells represented the majority of M. tuberculosis cells in the sputum of 21% of subjects with DS TB, and this proportion rose to 65% after 2 weeks of treatment with first-line drugs. In subjects with DR TB, DD- cells were found in the sputum of 29% of subjects prior to treatment initiation, and this proportion remained steady at 31% after 2 weeks of treatment with second-line drugs. By 2 months, DD- cells were detected in the sputum of only 2/15 (13.3%) subjects with DS TB and in 0/15 of subjects with DR TB. One of the DS subjects whose sputum was positive for DD- at month 2 later experienced treatment failure.