The Japanese eel Anguilla japonica is a catadromous fish, but it has recently been discovered that the use of freshwater habitat at the yellow eel stage is facultative. To determine if habitat use by Japanese eels differs between the sexes we examined the strontium:calcium (Sr:Ca) ratios in otoliths of 221 eels by electron probe micro-analyzer to reconstruct their environmental history. Eels were collected from the Kaoping River estuary of southwestern Taiwan from 1998 through 2005. The habitat use of yellow phase eels was divided into 3 types according to the life history pattern of the otolith Sr:Ca ratios: Type 1 (freshwater resident), Type 2 (brackish water resident with a freshwater preference) and Type 3 (brackish water resident with a seawater preference). Habitat use differed significantly between male and female silver stage eels. Females were classified predominantly as Type 2 or 3 while males were classified predominantly as Type 1 or 2. Consequently, female yellow stage eels preferred an estuarine habitat while males preferred a freshwater habitat. In addition, the mean otolith Sr:Ca ratios in the region 200 to 400 μm from the primordium (which corresponds to the period of sex differentiation) were higher in females than in males. This indicated that the sex differentiation of the eel might be related to habitat use, i.e. brackish water eels tended to differentiate as females and freshwater eels as males.