To quantify the salinity preference of juvenile green sturgeon Acipenser medirostris, two groups of A. medirostris [140 days post hatch (dph); total length (LT) 38·0–52·5 cm] were acclimated to either near fresh water (mean ± s.e. salinity = 3·2 ± 0·6) or full-strength salt water (34·1 ± 1·2) over 8 weeks. Following acclimation, the two groups were divided into experimental and control groups, where experimental A. medirostris from both freshwater and saltwater acclimations were individually introduced (200–220 dph) into a rectangular salinity-preference flume (maximum salinity gradient: 5–33). Control A. medirostris were presented with only their acclimation water (fresh water or salt water) on both sides of the flume. It was demonstrated that A. medirostris acclimated to both salt water and fresh water spent a significantly greater amount of time on the side of the testing area with the highest salinity concentration (P < 0·05 and P < 0·001, respectively) while control A. medirostris spent an equal amount of time on each side of the flume. These findings indicate that juvenile A. medirostris are not only capable of detecting salt water within the first year of their lives but perhaps are actively seeking out saline environments as they move through a watershed. Establishing A. medirostris salinity preferences provides a better understanding of the early life history of this threatened species, shedding light on possible outmigration timing.