Scrub typhus is a rickettsial disease transmitted to humans through the bite of chigger mites infected with Orientia tsutsugamushi, and is an endemic disease in Taiwan. To elucidate the molecular epidemiology of O. tsutsugamushi, the complete open reading frame of the 56-kDa type-specific antigen gene sequence of strains isolated from scrub typhus patients were determined and analyzed. A total of 116 isolates of O. tsutsugamushi were successfully isolated from patients infected in diverse geographic origins including Taiwan and three offshore islets, Kinmen, Matsu, and Penghu between May 2006 and December 2007. Sequence analysis revealed that 22 distinct sequence types could be identified that were broadly distributed in different clusters of the phylogenetic tree. Most of the isolates belong to Karp, Kawasaki, and Kuroki genotypes and are closely related to strains from Thailand, Japan, and Korea, whereas unique isolates different from other countries were also found in Taiwan. Distinct seasonal distributions were found in different sequence types. Some sequence types caused disease in the cold season, whereas others caused disease in the warm season.