BackgroundSeveral variables influence the serum concentration of thyroid hormones in dogs, including breed, age, drugs, and concurrent diseases. However, data regarding the interaction between thyroid function and the estrous stage of female dogs are limited.HypothesisEstrous stage may influence thyroid function in German Shepherd dogs.MethodsLongitudinal, observational, non‐randomized cohort study. The dogs were monitored during the complete estrous cycle, and different stages were determined by vaginal cytology. Two blood samples were collected at the beginning and end of each stage to analyze the following: total thyroxine (TT4), free thyroxine (fT4), total triiodothyronine (TT3), free triiodothyronine (fT3), canine thyrotropin (cTSH), progesterone, 17‐β‐estradiol, triglycerides, and cholesterol concentrations. Hematological and biochemical evaluations were performed at the beginning and end of the study period.AnimalsSeventeen German Shepherds were included, of which 7 were bred during the study period. One dog was excluded for estrus interruption and another for suspected hypothyroidism.ResultsSerum concentrations of T4, fT4, and fT3 were negatively correlated with age. Total thyroxine demonstrated significant changes in serum concentrations between estrous stages, with higher concentrations in estrus and diestrus. Total thyroxine concentrations were positively correlated with progesterone concentrations and negatively correlated with 17‐β‐estradiol concentrations. Free thyroxine did not show significant variations but was positively correlated with progesterone concentrations. Canine TSH concentrations were positively correlated with 17‐β‐estradiol concentrations. No significant differences in thyroid hormones and cTSH concentrations were observed between diestrus during pregnancy and pseudopregnancy.ConclusionsDifferent stages of estrus can influence the measurement of TT4 in female dogs.