In this study, iodine and thyroxin (T4) concentrations in the serum of 69 horses were investigated. Higher iodine concentrations were obtained from the horses housed in Chiba Prefecture. In contrast, T4 concentrations of horses at Shizuoka Prefecture were higher than those of horses at Chiba Prefecture. There was a significant correlation (r = 0.643, P < 0.001) between the iodine and T4 concentrations of horses at Saitama and Shizuoka prefectures. Although a significant correlation (r = 0.794, P < 0.001) was also observed in the investigation of all horses at Chiba Prefecture, the distribution area of the data was separated from the data of horses housed in Saitama and Shizuoka prefectures. A higher iodine concentration in the environment is expected in the sampling area at Chiba Prefecture. Thus, it was suggested that the concentrations of iodine in the serum of horses are influenced by geological differences. It was thought that equine serum is a useful sample for monitoring.