With technological improvements in otolith microchemical analysis, the flexible use of habitat from coastal marine to fresh waters has been discovered in Japanese eels Anguilla japonica. We examined the occurrence of 3 congeneric gill monogeneans-Pseudodactylogyrus anguillae, P. bini, and P. kamegaii-on wild Japanese eels, in relation to the host's flexibility. From April 2008 to October 2009, 114 eels were collected from a brackish-water cove and 2 rivers flowing into the cove in Ehime Prefecture, western Japan. Based on otolith microchemical analysis, the eels were discriminated according to the following 4 types of habitat use: freshwater residents (Type I), individuals utilizing low-salinity habitats (Type II), downstream habitat-shifters (Type III), and cove residents (Type IV). P. anguillae occurred mainly on Type I and II eels, while P. bini was primarily found on Type I eels. In contrast, P. kamegaii occurred mainly on Type III and IV eels. Thus, we conclude that species composition and infection levels of Pseudodactylogyrus spp. clearly differed with habitat-use patterns of Japanese eels. Also, since P. anguillae was scarcely found on either Type III or IV eels, this study suggests that previous identifications of monogeneans collected from European brackish-water localities as P. anguillae may require verification.