We investigated the variability of otolith chemistry in juvenile spotted seatrout from Chesapeake Bay seagrass habitats in 1998 and 2001, to assess whether otolith elemental and isotopic composition could be used to identify the most essential seagrass habitats for those juvenile fish. Otolith chemistry (Ca, Mn, Sr, Ba, and La; δ¹³C, δ¹⁸O) of juvenile fish collected in the five major seagrass habitats (Potomac, Rappahannock, York, Island, and Pocomoke Sound) showed significant variability within and between years. Although the ability of trace elements to allocate individual fish may vary between years, in combination with stable isotopes, they achieve high classification accuracy averaging 80–82% in the Pocomoke Sound and the Island, and 95–100% in the York and the Potomac habitats. The trace elements (Mn, Ba, and La) provided the best discrimination in 2001, a year of lower freshwater discharge than 1998. This is the first application of a rare earth element measured in otoliths (La) to discriminate habitats, and identify seagrass habitats for juvenile spotted seatrout at spatial scales of 15 km. Such fine spatial scale discrimination of habitats has not been previously achieved in estuaries and will distinguish fish born in individual seagrass beds in the Bay.