Chlamydia trachomatis is a global cause of blinding trachoma and sexually transmitted infections (STIs). We used comparative genomics of the family Chlamydiaceae to select conserved housekeeping genes for C. trachomatis multilocus sequencing, characterizing 19 reference and 68 clinical isolates from 6 continental/subcontinental regions. There were 44 sequence types (ST). Identical STs for STI isolates were recovered from different regions, whereas STs for trachoma isolates were restricted by continent. Twenty-nine of 52 alleles had nonuniform distributions of frequencies across regions (p<0.001). Phylogenetic analysis showed 3 disease clusters: invasive lymphogranuloma venereum strains, globally prevalent noninvasive STI strains (ompA genotypes D/Da, E, and F), and nonprevalent STI strains with a trachoma subcluster. Recombinant strains were observed among STI clusters. Single nucleotide polymorphisms (SNPs) were predictive of disease specificity. Multilocus and SNP typing can now be used to detect diverse and emerging C. trachomatis strains for epidemiologic and evolutionary studies of trachoma and STI populations worldwide.