The ontogeny of the cortisol stress response in yellow perch (Perca flavescens) was documented by measuring resting and stress-induced whole-body cortisol levels in fish at several early development stages. In newly fertilized eggs resting cortisol levels averaged 41.0 ± 3.0 pg/embryo, and decreased to 8.0 ± 3.0 pg/embryo by day 6 where they remained constant until hatch on day 10. Acute stress challenge tests (30 sec cold shock) conducted on embryonic (days 6 and 8 post-fertilization) and larval (hatch and 2 days post-hatch) yellow perch revealed no differences in whole-body cortisol levels between stressed and unstressed individuals. In acute stress challenge tests (30 sec net confinement) conducted on post-hatch yellow perch, differences in cortisol levels between stressed (30 min post-stress) and unstressed fish were detected one week after hatching (12.3 ± 1.6 ng/g and 7.0 ± 1.3 ng/g, respectively). The magnitude of the stress response was greater in 2- to 5-week-old fish than in one-week-old fish. Changes in post-stress cortisol levels were closely associated with changes in interrenal morphology. Cumulatively, these results indicate that cortisol biosynthesis, integration and maturation of the hypothalamic-pituitary-interrenal (HPI) axis can be observed in yellow perch as early as one week after hatching.