BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is considered to be less transmissible due to the fitness cost associated with drug resistance-conferring mutations in essential genes. OBJECTIVE: To test the hypothesis that TB drug resistance-conferring mutations with fitness cost are more frequent among human immunodeficiency virus (HIV) positive than among HIV-negative patients. DESIGN: We analysed all strains from the two TB drug resistance surveys conducted in Uganda between 2008 and 2011. Strains phenotypically susceptible to rifampicin and/or isoniazid were assumed to be wild-type; in all other cases, we performed whole-genome sequencing. Mutations at the rpoB531 and katG315 codons were considered without fitness loss, whereas other rpoB codons and non-katG were considered with fitness loss. RESULTS: Of the 897 TB patients, 286 (32.1%) were HIV-positive. Mutations with fitness loss in HIV-positive and HIV-negative patients were respectively as follows: non-531 rpoB: 1.03% (n = 3), 0.71% (n = 4) (OR 1.46, 95%CI 0.58-3.68); non-katG: 0.40% (n = 1), 1.0% (n = 6) (OR 0.40, 95%CI 0.07-2.20); rpoB531: 1.49% (n = 4), 0.69% (n = 4) (OR 2.29, 95%CI 0.83-5.77); katG315: 3.86% (n = 11), 2.55% (n = 15) (OR 1.54, 95%CI 0.81-2.90). The odds of mutations with and without fitness cost were higher for patients with a history of previous anti-tuberculosis treatment. CONCLUSIONS: Our data do not support the hypothesis that resistance-conferring mutations with fitness cost are likely to be often present in HIV-positive individuals.