This study was performed to determine the nationwide antigenic diversity of Orientia tsutsugamushi in South Korea. Sequence analysis was performed around variable domains I and II of a 56-kDa protein-encoding gene. We used eschar to overcome the disadvantages of conventional serotyping. The serological passive hemagglutination assay (PHA) was assessed based on the genotyping results. We analyzed 153 isolates from scrub typhus patients in major endemic areas and found that Boryong was the major strain (68.6%). New strains were also identified: Taguchi (19.6%), Kanda/Kawasaki (9.2%), and UAP7 (1.3%). PHA yielded significantly fewer positive results among Kawasaki strains (P < 0.001), which are not included in the PHA antigen panel. In South Korea, Boryong was still the predominant strain, but the sequence analysis identified new changes in minor strains (30.1%). This antigenic drift had a negative effect on the PHA results. Periodic surveillance of the contemporary strains using sequence analysis is needed.