The 22-kDa protein antigen is one of several antigens recognized by sera from scrub typhus patients infected with Orientia tsutsugamushi. The 22-kDa protein genes from six O. tsutsugamushi strains (Kato, TA763, AFSC 7, 18-032460, TH1814, MAK119) were cloned and their sequences were determined and compared to each other and to the Karp strain sequence listed in GenBank. The sequence alignment revealed that the promoter regions of these seven strains were highly conserved. However, the ORFs exhibited some variation. The phylogenetic analysis of the DNA sequences indicated that among the seven strains assessed, Kato and TA763 were the most closely related, while Karp and TH1814 were the most distantly related. The information gained from this analysis will facilitate our selection of O. tsutsugamushi strains from which antigens should be derived to be included in a multivalent vaccine candidate for scrub typhus.