Trace element and Sr-87/Sr-86 isotope analyses of fish pectoral fin rays offer non-destructive methods for determining habitat use. In this study, water and fin ray samples were analyzed for Gulf Sturgeon Acipenser oxyrinchus desotoi from the Choctawhatchee River Basin (FL and AL, USA) and compared with reference samples from Atlantic Sturgeon A. o. oxyrinchus held at controlled salinities (0, 10, 33 ppt). Samples were analyzed using inductively coupled plasma mass spectrometry, with a multi-collector for Sr-87/Sr-86. In water, Sr, Ba, Mn and Zn differed between freshwater and saline habitats, with increases in Sr and decreases in Ba, Mn and Zn. Sr-87/Sr-86 decreased upstream to downstream with lowest values in saline habitats. In the reference study, water trace element concentrations and Sr-87/Sr-86 corresponded to those in pectoral fin rays. Sr-87/Sr-86 was higher in pectoral fin ray than water, due to influence of diet, which differed with salinity. In wild fish, trace elements in pectoral fin rays indicated freshwater emigration to saline habitats primarily occurred in the second to third growth zone with some heterogeneity in the population (4% < 0.3 years, 39% 0.5-1.3 years, 39% 1.5-2.3 years, 17% 2.5-3.3 years). Analyses of Sr-87/Sr-86 indicated initial locations of Gulf Sturgeon were in the middle river, with few fish in the upper or lower river. Most (74%) juvenile Gulf Sturgeon utilized more than one river region prior to freshwater emigration and 48% moved upstream temporarily based on increased Sr-87/Sr-86. After initial freshwater emigration, fish utilized lower-river to saline habitats. Collectively, these studies demonstrate the usefulness of trace element and Sr-87/Sr-86 analyses in sturgeon pectoral fin rays.