Tuberculosis (TB) is one of the most common opportunistic diseases that appear among human immunodeficiency virus (HIV)-positive patients in Haiti. In this context the probable emergence of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis is of great epidemiological concern. However, as routine culture of M. tuberculosis and drug susceptibility testing are not performed in Haiti, it has not been possible so far to evaluate the rate of drug resistance among M. tuberculosis isolates from circulating TB cases. This report describes the first study on the molecular typing and drug resistance of M. tuberculosis isolates from patients with culture-positive pulmonary tuberculosis monitored at the GHESKIO Centers in Haiti during the year 2000. Clinical, epidemiological, and drug susceptibility testing results were available for 157 patients with confirmed cases of TB, with a total of 8.9% of patients harboring MDR M. tuberculosis. A significant association between the occurrence of resistance and previous TB treatment was observed (P < 0.001), suggesting that a previous history of TB treatment was a risk factor associated with MDR TB in Haiti. The DNAs of individual isolates from 106 samples were available and were typed by spoligotyping and determination of the variable number of tandem DNA repeats. Both typing methods provided interpretable results for 96 isolates, and the clusters observed were further confirmed by ligation-mediated PCR to define potential cases of active transmission. Thirty-three (34%) of the isolates were found to be grouped into 11 clusters with two or more identical patterns. However, an assessment of risk factors (sex, HIV positivity, previous treatment, drug resistance) showed that none was significantly associated with the active transmission of TB. These observations suggest that acquired MDR TB is prevalent in Haiti and may be associated with compliance issues during TB treatment since prior TB therapy is the strongest risk factor associated with MDR TB. Prevention of TB transmission in Haiti should target active case investigation, routine detection of drug resistance, and adequate treatment of patients. The use of directly observed short-course therapy should be enforced throughout the country; and relapses, reactivations, or newly acquired infections should be discriminated by genotyping methods.