Comparison of the pattern of synonymous nucleotide substitution between two complete genomes of Mycobacterium tuberculosis at 3,298 putatively orthologous loci showed a mean percent difference per synonymous site of 0.000328±0.000022. Although 80.5% of loci showed no synonymous or nonsynonymous nucleotide differences, the level of polymorphism observed at other loci was greater than suggested by previous studies of a small number of loci. This level of nucleotide difference leads to the conservative estimate that the common ancestor of these two genotypes occurred approximately 35,000 ago, which is twice as high as some recent estimates of the time of origin of this species. Our results suggest that a large number of loci should be examined for an accurate assessment of the level of nucleotide diversity in natural populations of pathogenic microorganisms.