Thyroid hormones and the factors influencing them are rarely studied in tortoises. This study therefore aimed to calculate reference intervals (RI) for thyroid hormones and iodine concentrations by species, sexes, and seasons in four species of adult Mediterranean tortoises (Testudo spp.) and to evaluate possible correlations between plasma thyroid hormones, serum iodine, plasma protein, and increased liver and kidney values. Thyroid hormones (total tetraiodothyronine [tT4], free tetraiodothyronine [fT4], total triiodothyronine [tT3], and free triiodothyronine [fT3]) were measured in plasma from adult, healthy Hermann's (Testudo hermanni, n = 255), spur-thighed (Testudo graeca, n = 89), marginated (Testudo marginata, n = 72), and Russian tortoises (Testudo horsfieldii, n = 30). Species, sex, and seasonal variations were determined by Bayesian information criterion (BIC), and correlations between thyroid hormones, protein, iodine, and increased liver/kidney values were evaluated by Spearman's rank correlation test. Total T4 did not reveal any species, sex, or seasonal differences (RI: 0.102–0.455 µg/dl), while seasonal differences were found for fT4 (RI: spring 0.624–9.012; summer 0.379–5.476; fall 0.376–5.426 pmol/L). The tT3 concentrations differed significantly depending on species, season, and the interaction of species × season. Seasonal differences were also found for fT3 and iodine. Several significant (P ≤ 0.05) correlations were detected between the analytes, including a positive correlation between tT4 and fT4. These results provide a tool for the evaluation of thyroid hormone concentrations in Mediterranean tortoises and confirm the influence of season on thyroid function in these animals.