Context: Skeletal muscle mass (SMM) is one of the major components of human body composition, with deviations from normal values often leading to sarcopenia. Objective: Our major aim was to conduct a genome-wide DNA methylation study in an attempt to identify potential genomic regions associated with SMM. Design: This was a mixed cross-sectional and longitudinal study. Setting: Community-based study. Participants: A total of 1550 middle-aged United Kingdom twins (monozygotic [MZ] and dizygotic [DZ]), 297 of which were repeatedly measured participated in the study. Main Outcome Measure: Appendicular lean mass assessed using dual-energy X-ray absorptiometry technology, and methylated DNA immunoprecipitation sequencing DNA methylation profiling genome-wide were obtained from each individual. Results: Heritability estimate of SMM, with simultaneous adjustment for covariates obtained using variance decomposition analysis, was h2= 0.809 ± 0.050. After quality control and analysis of longitudinal stability, the DNA methylation data comprised of 723 029 genomic sites, with positive correlations between repeated measurements (Rrepeated=0.114-0.905). Correlations between MZ and DZ twins were 0.51 and 0.38 at a genome-wide average, respectively, and clearly increased with Rrepeated. Testing for DNA methylation association with SMM in 50 discordant MZ twins revealed 36 081 nominally significant results, of which the top-ranked 134 signals (P≤.01 and Rrepeated ≥0.40) were subjected to replication in the sample of 1196 individuals. SevenSMMmethylation association signals replicated at a false discovery rate less than 0.1, and these were located in or near genes DNAH12, CAND1, CYP4F29P, and ZFP64, which have previously been highlighted in muscle-related studies. Adjusting for age, smoking, and blood cell heterogeneity did not alter significance of these associations. Conclusion: This epigenome-wide study, testing longitudinally stable methylation sites, discovered and replicated a number of associations between DNA methylation at CpG loci and SMM. Four replicated signals were related to genes with potential muscle functions, suggesting that the methylome of whole blood may be informative ofSMMvariation.