Thyroid hormones play a critical physiologic role in regulating protein synthesis, growth, and metabolism. To date, because no published compilation of baseline values for thyroid hormones in beluga whales (Delphinapterus leucas) exists, assessment of thyroid hormone concentrations in this species has been underused in clinical settings. The purpose of this study was to document the concentrations of total thyroxine (tT4) and total triiodothyronine (tT3) in healthy aquarium-maintained and free-ranging beluga whales and to determine the influence of age, sex, and season on the thyroid hormone concentrations. Archived serum samples were collected from healthy aquarium-maintained (n = 43) and free-ranging (n = 39) belugas, and serum tT4 and tT3 were measured using chemiluminescence immunoassay. The mean tT4 concentration in aquarium-maintained belugas was 5.67 ± 1.43 μg/dl and the mean tT3 concentration was 70.72 ± 2.37 ng/dl. Sex comparisons showed that aquarium-maintained males had significantly greater tT4 and tT3 (9.70 ± 4.48 μg/dl and 92.65 ± 30.55 ng/dl, respectively) than females (7.18 ± 2.82 μg/dl and 77.95 ± 20.37 ng/dl) (P = 0.004 and P = 0.013). Age comparisons showed that aquarium-maintained whales aged 1–5 yr had the highest concentrations of tT4 and tT3 (8.17 ± 0.17 μg/dl and 105.46 ± 1.98 ng/dl, respectively) (P = 0.002 and P < 0.001). tT4 concentrations differed significantly between seasons, with concentrations in winter (4.59 ± 1.09 μg/dl) being significantly decreased compared with spring (P = 0.009), summer (P < 0.0001), and fall (P < 0.0001) concentrations. There was a significant difference in tT4 and tT3 concentrations between aquarium-maintained whales (5.67 ± 1.43 μg/dl and 70.72 ± 15.57 ng/dl, respectively) and free-ranging whales (11.71 ± 3.36 μg/dl and 103.38 ± 26.45 ng/dl) (P < 0.0001 and P < 0.001). Clinicians should consider biologic and environmental influences (age, sex, and season) for a more accurate interpretation of thyroid hormone concentrations in belugas. The findings of this study provide a baseline for thyroid health monitoring and comprehensive health assessments in both aquarium-maintained and free-ranging beluga whales.