Knowledge of subspecies () outer membrane protein (OMP) sequence variability is essential for understanding spirochete proliferation within endemic populations as well as the design of a globally effective syphilis vaccine. Our group has identified extracellular loops (ECLs) of BamA (TP0326) and members of the FadL family (TP0548, TP0856, TP0858, TP0859, and TP0865) as potential components of a multivalent vaccine cocktail. As part of a consortium to explore strain diversity, we mapped the variability of BamA and FadL orthologs in 186 strains from Malawi, China, and Colombia onto predicted 3D structures. The 186 genomes fell into eight subclades (five Nichols- and three SS14-lineage) with substantial geographic restriction. Single nucleotide variants accounted for the large majority of proteoforms, with variability notably higher within the Nichols-lineage strains. Most mutations were in regions of the proteins predicted to be extracellular and harboring B cell epitopes. We observed a striking difference in the degree of variability between the six OMPs, suggesting that these proteins are following divergent evolutionary paths. Concatenation of OMP sequences recapitulated the phylogenetic structure of the strains, effectively segregating within clades and largely clustering by subclades. Finally, we noted that BamA and FadL candidate ECL vaccinogens, previously shown to elicit antibodies that kill treponemes during cultivation, are well conserved. Taken as a whole, our study establishes a structural-phylogenetic approach for analyzing the forces shaping the host-pathogen interface in syphilis within endemic populations while informing the selection of vaccine targets.IMPORTANCESyphilis remains a major global health concern, reinforcing the need for a safe and effective vaccine. Understanding the variability of OMPs is essential for tracking pathogen evolution and informing vaccine design. Here, we analyzed the variability of six OMPs in 186 strains from Malawi, China, and Colombia, identifying protein-specific evolutionary patterns. Most mutations were localized in extracellular regions and, notably, appeared to correlate with the phylogenetic structure of . Despite OMP heterogeneity, several candidate vaccinogens remained highly conserved, reinforcing their potential as globally effective vaccine targets. Our study establishes a structural-phylogenetic framework for dissecting the forces shaping the host-spirochete interface within endemic populations and provides a foundation for designing a globally effective syphilis vaccine.