The purpose of this study was to establish physiologic reference values for intraocular pressure (IOP) in clinically healthy trout (Salvelinus fontinalis). The study population consisted of 15 adult brook trout (14 male, 1 female); body weight ranged from 0.47 to 1.15 kg; length from 33.5 to 45.2 cm; and eye diameter from 11 to 14 mm. Animals were anesthetized, and a preliminary ophthalmic exam was performed. IOP was then measured by rebound tonometer. Three values were obtained for each eye. The animals were subsequently euthanized, and postmortem IOP was measured by the same method. A standard necropsy was performed on each fish to determine sex and to examine it for gross lesions. Premortem results (mm Hg) of measurements in the right eyes (n = 15) were 8.82 ± 2.45 mm Hg (minimum–maximum [min/max], 5–14 mm Hg; median, 8.33 mm Hg), and postmortem results were 6.31 ± 2.31 mm Hg (min/max, 3–11 mm Hg; median, 6 mmHg). Premortem results (mm Hg) of measurements in the left eyes (n = 15) were 9.20 ± 1.91 mm Hg (min/max, 5.33–12.33 mm Hg; median, 10 mm Hg), and postmortem results were and 9.81 ± 2.19mm Hg (min/max, 5–13 mmHg; median, 10.17mm Hg). A Mann-Whitney U-test was performed and revealed no significant difference within an eye under both pre- and postmortem conditions, and no significant difference between eyes between pre- and postmortem conditions. Rebound tonometry is a practical and efficient means of determining IOP in freshly euthanized fish or in anesthetized fish, where it requires minimal anesthetic and restraint time; it could potentially serve as a useful diagnostic tool when evaluating ophthalmic health.