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Objectives: The objective of the study was to investigate the prevalence of hyperthyroidism in Spain, including its geographical distribution and prevalence across different age groups. Methods: A cross-sectional study was conducted. Samples submitted to a reference laboratory to evaluate serum total thyroxine concentration (TT4) during a 3-year period were evaluated (n = 27,888). A cat was considered hyperthyroid if the TT4 concentration was greater than 60.4 nmol/l (4.7 μg/dl). Hyperthyroid cats were classified based on their TT4 concentration at diagnosis as mildly (TT4 60.4–124.8 nmol/l [4.7–9.7 μg/dl]), moderately (TT4 124.8–249.68 nmol/l [9.7–19.4 μg/dl]) or severely (TT4 >249.6 nmol/l [>19.4 μg/dl]) hyperthyroid. The samples were also grouped by life stage, into young adults (1–6 years old), mature adults (7–10 years old) and seniors (>10 years old). The study included samples from 17 regions of Spain and geographical mapping was based on seven domains. Results: One thousand seven hundred and twenty-two out of 27,888 cats (6.2%, 95% confidence interval [CI] = 5.9–6.5) were classified as hyperthyroid. Within the hyperthyroid group, ages ranged from 2 to 25 years old, with a median of 14 years. Within the hyperthyroid population, 14/368 (3.8%), 27/368 (7.3%) and 327/368 (88.8%) were young adults, mature adults and seniors, respectively. The prevalence of hyperthyroidism in this laboratory-based sample in Spain was 2.7 (95% CI = 0.4–5.7) to 6.9% (95% CI = 6.4–7.4), depending on the geographical region. During the study period, 8.2% of cases that were initially non-hyperthyroid were later diagnosed with hyperthyroidism. Conclusions and relevance: Prevalence of hyperthyroidism in Spain is 6.2% (95% CI = 5.9–6.5) overall and 7.9% (95% CI = 7.2–8.8) in cats older than 10 years. The distribution of the disease is heterogeneous between different geographical regions. Further large-scale prospective studies are required to determine the risk factors in each region and understand the reasons for this variation. [ABSTRACT FROM AUTHOR]