Habitat use of the tapertail anchovy (Coilia mystus Linnaeus, 1758) from the Oujiang River Estuary and the Zhujiang (Pearl) River Estuary was studied by examining the environmental signatures of Sr and Ca in otoliths using electron probe microanalysis. Individuals from the Oujiang River had higher and varied Sr:Ca ratios (expressed as (Sr:Ca)×1 000, 3.83–13.0 average) in the otolith core regions, suggesting that they were born in brackish or sea waters, and that a freshwater habitat might not be necessary for egg hatching and larval growth. While, individuals from the Zhujiang River had lower Sr:Ca ratios (0.39–2.51 average) in the core regions, suggesting a freshwater origin. After hatching, anchovies from the Zhujiang River migrate downstream to the river estuary close to brackish water. Our results demonstrated varied habitat use for spawning during stages of early life history between the two populations, and suggested that such variations are promoting diversity of life history strategies of this species.