ABSTRACTTprK antigenic variation is acknowledged as an important strategy developed byTreponema pallidumto achieve immune evasion. Previous studies applied short-read sequencing to exploretprKgene sequence diversity in clinical samples; however, due to the limitations of short-read sequencing, it was difficult to determine the linkage between the seven V regions, and crucial information about full-lengthtprKvariants was lost. Although two recent studies explored completetprKgene profiles in natural human syphilis infection, there are still too few profiled full-lengthtprKvariants among clinicalT. pallidumisolates to fully understand the characteristics of TprK coding diversity. Here, Pacific Biosciences (PacBio) long-read sequencing was applied to examine the diversity of full-lengthtprKvariants in 21 clinicalT. pallidumisolates from 11 patients with primary syphilis and 10 patients with secondary syphilis. A total of 398 high-confidence full-length sequences, which presented remarkable sequence heterogeneity, were found. However, these full-lengthtprKvariants exhibited limited variation in length and GC content, showing 24 length types and average GC content of 51.5 ± 0.42% and 51.6 ± 0.26% for primary and secondary syphilis samples, respectively. Additionally, the combined patterns of mutated V regions generating newtprKvariants were obviously different in primary and secondary syphilis samples. The diversity oftprKgene sequences in primary syphilis samples may represent the underlying variability of the bacterium; conversely, the variability of thetprKgene in secondary syphilis samples may more accurately reflect howT. pallidumescapes host immune clearance. These data highlight thetprKgene as an important coding gene that shows conflicting genetic characteristics but underlies the persistence of spirochete infection.IMPORTANCEThe resurgence of syphilis in both low- and high-income countries has attracted attention, and persistent infection by the pathogen has long been a research focus. ThetprKgene, encoding the hypervariable outer membrane protein, is thought to be responsible for pathogen immune evasion and persistent infection. Here, PacBio long-read sequencing was applied to examine the diversity of full-lengthtprKvariants in 21 clinicalT. pallidumisolates from 11 patients with primary syphilis and 10 patients with secondary syphilis. The results showed that the sequences of thetprKgene were remarkably heterogeneous; however, the sequences presented limited variation in length and GC content. The investigation of the combined patterns of the V regions allowed us to gain insight into the features of thetprKgene generating new variants at different clinical stages. The findings of this study will be helpful for further exploration of the pathogenesis of syphilis.