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SUMMARY SETTING Although approximately 0.5 million cases of multidrug-resistant tuberculosis (MDR-TB) occur globally each year, surveillance data are limited. Botswana is one of the few high TB burden countries to have carried out multiple anti-tuberculosis drug resistance surveys (in 1995–1996, 1999 and 2002). OBJECTIVE In 2007–2008, we conducted the fourth national survey of anti-tuberculosis drug resistance in Botswana to assess anti-tuberculosis drug resistance, including trends over time. In the previous survey, 0.8% (95%CI 0.4–1.5) of new patients and 10.4% (95%CI 5.6–17.3) of previously treated patients had MDR-TB. DESIGN During the survey period, eligible specimens from all new sputum-smear positive TB patients and from all TB patients with history of previous anti-tuberculosis treatment underwent mycobacterial culture and anti-tuberculosis drug susceptibility testing (DST). RESULTS Of 924 new TB patients and 137 with previous anti-tuberculosis treatment with DST results, respectively 23 (2.5%, 95%CI 1.6–3.7) and 9 (6.6%, 95%CI 3.3–11.7) had MDR-TB. The proportion of new TB patients with MDR-TB has tripled in Botswana since the previous survey. CONCLUSION Combatting drug-resistant TB will require the scale-up of MDR-TB diagnosis and treatment to prevent the transmission of MDR-TB and strengthening of general TB control to prevent the emergence of resistance.