AIT (autoimmune thyroiditis) is a complex disease influenced by genetic and environmental factors as well as immune dysregulation. Epigenetics has unveiled potential connections among environmental factors, gene expression and thyroid autoimmunity. Among epigenetic modifications, DNA methylation is the first discovered and the most extensively-studied. Investigations both domestically and internationally indicate that iodine supplementation in areas with either excessive or insufficient iodine levels increases the incidence of AIT. Chemokines also play a crucial role in the pathogenesis of AIT. Therefore, does iodine influence the DNA methylation of chemokine genes of patients with AIT, and what are the potential mechanisms involved?. Healthy controls and patients with AIT were matched at the ratio of 1:1 according to age, sex, BMI and residential address, and a total of 176 patients with AIT together with 176 controls were included from regions with varying iodine levels. DNA methylation and mRNA expression levels were analyzed in whole blood using MethylTarget and qRT-PCR methods. At the same time, the GSE138198 and GSE54958 datasets were downloaded from GEO to obtain transcriptional datasets of thyroid tissues from patients with AIT. AIT patients had lower DNA methylation levels in CCL5_2 and CXCL8_1 target regions than controls, while the mRNA expression of CCL5 and CXCL8 genes was significantly higher. A negative correlation was found between the DNA methylation of CCL5_2 and its CpG sites as well as CCL5 gene expression. Higher CCL5 mRNA expression was validated in the thyroid tissues of patients with AIT using GSE datasets. DNA methylation differences at different iodine levels were mainly observed in CCL5_1, CCL5_2, CXCL8_1 and CXCR5_1. CXCL8_1 showed a positive correlation with UIC (urinary iodine concentration). This study demonstrates an association between the DNA methylation status of CCL5 and CXCL8 genes and AIT. The DNA methylation level of the CCL5 gene can serve as an epigenetic marker and biological indicator for AIT. Additionally, long-term iodine deficiency supplementation has a more pronounced impact on the DNA methylation levels of CCL5 and CXCL8 genes.