BACKGROUND Reference intervals for thyroid hormones (TH) concentrations have not been previously established for donkeys, leading to potential misdiagnosis of thyroid disease. OBJECTIVE To determine the normal values of TH in healthy adult donkeys and compare them to TH values from healthy adult horses. METHODS Thirty-eight healthy Andalusian donkeys and 19 healthy Andalusian horses from 2 different farms were used. Donkeys were divided into 3 age groups: <5, 5-10 and >11 years and into 2 gender groups. Serum concentrations of fT3, tT3, rT3, fT4 and tT4 were quantified by radioimmunoassay. All blood samples were collected the same day in the morning. None of the animals had received any treatment for 30 days prior to sampling or had any history of disease. Both farms were in close proximity and under similar management. Differences between groups were determined using a one-way ANOVA analysis followed by Fisher's LSD test. P<0.05 was considered significant. RESULTS Serum TH concentrations were higher in donkeys than in horses (P<0.01). Donkeys <5 years had higher serum rT3, fT4 and tT4 concentrations than donkeys >5 years (P<0.05). Furthermore, older donkeys (>11 years) had lower serum fT3 and tT3 concentrations than younger donkeys' groups (<5 and 5-10 years, P<0.05). TH concentrations were not different between genders (fT3: P = 0.06; tT3: P = 0.08; rT3: P = 0.15; fT4: P = 0.89; and tT4: P = 0.19). CONCLUSIONS Thyroid hormone concentrations are different between healthy adult donkeys and horses. CONCLUSIONS Establishing species-specific TH reference ranges is important when evaluating clinicopathologic data in equids in order to avoid the misdiagnosis of thyroid gland dysfunction. Further studies to elucidate the physiological mechanisms leading to these differences are warranted.