This study aimed to reconstruct the evolutionary history of Beijing strains of Mycobacterium tuberculosis and to test the hypothesis that evolution has influenced the ability of the Beijing strains within the different Beijing sublineages to spread and cause disease. A PCR-based method was used to analyze the genome structure of 40 different loci in 325 Beijing isolates collected from new and retreatment tuberculosis patients from an urban setting and 270 Beijing isolates collected from high-risk tuberculosis patients from a rural setting in the Western Cape, South Africa. The resulting data were subjected to phylogenetic analysis using the neighbor joining algorithm. Phylogenetic reconstructions were highly congruent with the "gold standard" phylogenetic tree based on synonymous single-nucleotide polymorphisms, thereby allowing a prediction of the order in which the evolutionary events had occurred. A total of seven independently evolving Beijing sublineages were identified. Analysis of epidemiological data in relation to the Beijing sublineage suggested an association between recent evolutionary change and frequency of occurrence in an urban population (P<0.001) as well as in the rural population (P<0.001). This concept was further supported by an association between more recently evolved Beijing strains and an increased ability to transmit and to cause disease (odds ratio, 5.82; 95% confidence interval, 3.13 to 10.82 [P<0.001]). An association between Beijing sublineage and demographic and clinical parameters and drug resistance could not be demonstrated. From these data, we suggest that the pathogenic characteristics of Beijing strains are not conserved but rather that strains within individual lineages have evolved unique pathogenic characteristics.