The genetic diversity in 502 isolates of Mycobacterium tuberculosis from the Eastern Cape Province of South Africa was DNA-profiled using random amplified polymorphic DNA sequences generated by four primers, and analyzed using the Dice similarity coefficient and the Ward cluster algorithm. three major cluster groups and ten clusters were detected and shown to be significant at the five percent level using analysis of molecular variance (AMOVA). It is proposed that the three major cluster groups may represent three different entries of M. tuberculosis into South Africa during colonial times. No significant genetic differences could be detected between the urban and rural populations, suggesting that migration may pray an important role in the wide transmission of all strains of the organism found in this province. However, a genetic distinction (p = 0.10) could be detected between rural individuals from the southern, more prosperous, region and the northern, less prosperous, regions of the province, which may represent either a limited migration between distant rural communities or a difference in health treatment historically in the two areas. AMOVA indicates the existence of a higher degree of population structure between drug sensitive and drug-resistant individuals than between urban and rural individuals (p = 0.05). Two clusters, C1 and C3, contained a large proportion of the isolates resistant to between one and three first-line drugs. isolates resistant to more than three antibiotics were genetically diverse, suggesting that they are as yet not being transmitted at a high rate between individuals.