The epidemiology of extensively drug-resistant tuberculosis (XDR-TB), an emerging threat to TB control, is not well understood.To gain insight into the genotypic population structure of XDR Mycobacterium tuberculosis strains in South Africa using a molecular approach and thereby determine whether XDR-TB is mainly acquired or transmitted.Sputum isolates from patients with multidrug-resistant tuberculosis (MDR-TB) were submitted to the National Referral Laboratory for second-line drug susceptibility testing. The XDR-TB isolates were spoligotyped and these data were compared to the geographic origin of the isolate.Of the 699 MDR-TB isolates submitted for testing between June 2005 and December 2006, 101 (17%) patients had a culture that was resistant to either ofloxacin or kanamycin, and 41 (6%) were resistant to both drugs (XDR-TB). Spoligotyping of the XDR-TB isolates identified 17 genotypes. As a result of the high genotypic diversity and geographical distribution, we estimate that between 63% and 75% of cases developed XDR-TB through acquisition.Acquisition of extensive drug resistance appears to be the primary mechanism driving the XDR-TB epidemic in South Africa. This urgent TB control issue has to be addressed to prevent the spread of this potentially incurable disease.