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The aim of this study was to investigate the reproducibility of serum tests for total thyroxine (T4) in three German veterinary laboratories. Serum samples from 53 dogs with clinically suspected hypothyroidism were examined for this purpose. The serum samples were divided into 5 aliquots, labeled with different names, and sent to the respective laboratories. The agreement of the values between the three laboratories (within or below the respective reference range) was evaluated. In 23/41 dogs, the values were uniformly below, within, or above the respective reference range in all laboratories. The agreement between laboratories 1 and 2 and laboratories 1 and 3 was low ( = 0.30 and 0.25, respectively), while there was a high degree of agreement between laboratories 2 and 3 ( = 0.68). The variable agreement in the evaluation of thyroxine concentrations in the same serum sample by different laboratories or at different evaluation times emphasizes the high relevance of interpretingsuch values in the context of the symptoms present and other laboratory values such as TSH concentration.