Fish hatcheries are generally not adjacent to stocking locations. Thus, hatchery fish undergo transportation processes for variable durations that can result in changes in water quality, fish physiology (e.g., whole blood glucose and plasma cortisol), and reduced survival. Walleye Sander vitreus are commonly stocked throughout North America with variable stocking success, possibly due to altered physiological responses associated with changes in water quality parameters during transportation. We hypothesized increased transport duration would be associated with increases in water temperature, carbon dioxide, and total ammonia nitrogen and decreases in pH and total alkalinity. We also hypothesized that increases in carbon dioxide, water temperature, un-ionized ammonia, and total ammonia nitrogen would be positively related with Walleye whole blood glucose and plasma cortisol concentrations. Walleye were transported for either 0, 0.5, 3, or 5 h and whole blood glucose and plasma cortisol concentrations and mortality were evaluated for 48 h posttransport. Total ammonia nitrogen concentrations, carbon dioxide, pH, and water temperature increased with transportation duration while total alkalinity decreased. Plasma cortisol and whole blood glucose concentrations of Walleye transported for longer durations took longer to decline relative to those not transported. Water quality parameters were not associated with changes in Walleye whole blood glucose and plasma cortisol concentrations, but they were negatively related with time since transport (P < 0.05). Despite increases in stress, mortality was low (2.5%). Overall, we found evidence to support our hypotheses regarding reduced water quality associated with increasing transport duration. Finally, whole blood glucose and plasma cortisol concentration of Walleye that were transported were similar to those not transported, suggesting handling procedures before transportation could play a significant role in physiological responses measured after transportation. Further, evaluation of stocking procedures aimed at decreasing handling during the loading process could enhance stocking protocols. Elevated concentrations of whole blood glucose and plasma cortisol following transport could make recently stocked Walleye more susceptible to the effects of other environmental factors such as starvation, predation, and disease, all of which could result in increased mortality rates after stocking.