The Nisqually Fall Chinook salmon (Oncorhynchus tshawytscha) population is one of 27 stocks in the Puget Sound (Washington) evolutionarily significant unit listed as threatened under the federal Endangered Species Act (ESA). Extensive restoration of the Nisqually River delta ecosystem has taken place to assist in recovery of the stock since estuary habitat is a critical transition zone for juvenile fall Chinook salmon. A pre-restoration baseline that includes the characterization of life history strategies, estuary residence times, growth rates and habitat use is needed to evaluate the potential response of hatchery and natural origin Chinook salmon to restoration efforts and to determine restoration success. Otolith microstructure analysis was selected as a tool to examine Chinook salmon life history, growth and residence in the Nisqually River estuary. The purpose of the current study is to incorporate microstructural analysis from the otoliths of juvenile Nisqually Chinook salmon collected at the downstream migrant trap within true freshwater (FW) habitat of the Nisqually River. The results from this analysis confirmed the previously documented Nisqually-specific FW microstructure pattern and revealed a Nisqually-specific microstructure pattern early in development (“developmental pattern”). No inter-annual variation in the microstructure pattern was visually observed when compared to samples from previous years. Furthermore, the Nisqually-specific “developmental pattern” and the FW microstructure pattern used in combination during analysis will allow us to recognize and separate with further confidence future unmarked Chinook salmon otolith collections into Nisqually-origin (natural or unmarked hatchery) and non-Nisqually origin categories. Freshwater mean increment width, growth rate and residence time were also calculated.