The sexual differentiation in blood biochemical values were assessed in rainbow trout (Oncorhynchus mykiss) cultured in Laribal Trout Culture Farm, Dachigam National Park, Srinagar, India. The blood from the healthy rainbow trout brood was collected for serum and analyzed & expressed as mean±SD in male and female fish. The present investigation revealed some significantly higher in males than female fish (P≤0.05), including total protein (6.8±2.1, 4.4±0.8 g/dl), albumin (0.52±0.1, 0.35±0.2 mg/dl), glucose (5.8±0.3, 4.14±0.5 mg/dl), phosphorous (1.5±0.1, 1.4±0.1 mg/dl), magnesium (4.2±0.3, 3.7±0.2 mg/dl). There were no significant differences with higher values in male than female of the C3 (39.2±3.7, 37.3±3.6 mg/dl), C4 (26.3±2.5, 36.1±3.4 mg/dl), alkaline phosphatase (23.2±2.5, 19.8±2.3 IU/L), amylase (161.4±22.4, 149.2±11.8 IU/L), cholesterol (54.2±2.8, 48.5±2.8 mg/dl) and creatinine phosphokinase (55.2±3.4, 49.8±2.8 IU/L). The other biochemical parameters were higher in female and the differences were not significant, in case of aspartate aminotransferase (70.3±7.2, 88.3±4.8 IU/L), alanine aminotransferase (21.9±3.6, 29.6±6.2 IU/L), IgM (80.1±8.2, 92.1±8.4 mg/dl), Iron (25.4±2.1, 29.2±0.5 µg/dl), calcium (6.9±0.2, 6.8±0.3 mg/dl). The findings would be helpful to establish a baseline to draw any conclusive remarks against the health status of rainbow trout, showing deviation from the normal parameters.