Tobacco smoking is a preventable environmental factor that contributes to a wide spectrum of age-related health outcomes; however, its association with the development of frailty is not yet well established. We examined the associations of self-reported smoking indicators, serum cotinine levels and smoking-related DNA methylation biomarkers with a quantitative frailty index (FI) in 2 independent subsets of older adults (age 50-75) recruited in Saarland, Germany in 2000 - 2002 (discovery set: n = 978, validation set: n = 531). We obtained DNA methylation profiles in whole blood samples by Illumina HumanMethylation450 BeadChip and calculated the FI according to the method of Mitnitski and Rockwood. Mixed linear regression models were implemented to assess the associations between smoking indicators and the FI. After controlling for potential covariates, current smoking, cumulative smoking exposure (pack-years), and time after smoking cessation (years) were significantly associated with the FI (P-value < 0.05). In the discovery panel, 17 out of 151 previously identified smoking-related CpG sites were associated with the FI after correction for multiple testing (FDR < 0.05). Nine of them survived in the validation phase and were designated as frailty-associated loci. A smoking index (SI) based on the 9 loci manifested a monotonic association with the FI. In conclusion, this study suggested that epigenetic alterations could play a role in smoking-associated development of frailty. The identified CpG sites have the potential to be prognostic biomarkers of frailty and frailty-related health outcomes. Our findings and the underlying mechanisms should be followed up in further, preferably longitudinal studies.