This study was conducted to determine plasma thyroxine (T4) concentrations in zoo-kept black-tailed prairie dogs (Cynomys ludovicianus). Thirty-one healthy prairie dogs of both sexes were studied as part of their annual clinical health evaluation, performed under general isoflurane anesthesia. Each animal underwent a complete physical examination, complete blood count, plasma biochemistry, and venous blood gas analysis. Heparinized venous blood samples were collected individually and processed for plasma T4 analysis using a veterinary biochemistry analyzer. The median plasma T4 concentration for the prairie dogs in this study was 4.1 μg/dl (minimum = 0.6 μg/dl; maximum = 8.0 μg/dl). The mean ± standard deviation plasma T4 concentration was 4.49 ± 2.39 μg/dl. No significant differences were found for varying ages, sexes, weights, or housing systems. The data presented in this report can promote better physiologic understanding and improve clinical management of this rodent species.