ObjectiveTo explore the differences in DNA methylation associated with age-related hearing loss in a study of 57 twin pairs from China. DesignMonozygotic twins were identified through the Qingdao Twin Registration system. The median age of participants was > 50 years. Their hearing thresholds were measured using a multilevel pure-tone audiometry assessment. The pure-tone audiometry was calculated at low frequencies (0.5, 1.0, and 2.0 kHz), speech frequencies (0.5, 1.0, 2.0, and 4.0 kHz), and high frequencies (4.0 and 8 kHz). The CpG sites were tested using a linear mixed-effects model, and the function of the cis-regulatory regions and ontological enrichments were predicted using the online Genomic Regions Enrichment of Annotations Tool. The differentially methylated regions were identified using a comb-p python library approach. ResultsIn each of the PTA categories (low-, speech-, high-frequency), age-related hearing loss was detected in 25.9%, 19.3%, and 52.8% of participants. In the low-, speech- and high-frequency categories we identified 18, 42, and 12 individual CpG sites and 6, 11, and 6 differentially methylated regions. The CpG site located near DUSP4 had the strongest association with low- and speech-frequency, while the strongest association with high-frequency was near C21orf58. We identified associations of ALG10 with high-frequency hearing, C3 and LCK with low- and speech-frequency hearing, and GBX2 with low-frequency hearing. Top pathways that may be related to hearing, such as the Notch signaling pathway, were also identified. ConclusionOur study is the first of its kind to identify these genes and their associated with DNA methylation may play essential roles in the hearing process. The results of our epigenome-wide association study on twins clarify the complex mechanisms underlying age-related hearing loss.