The circadian clock system instructs 24-h rhythmicity on gene expression in essentially all cells, including adipocytes, and epigenetic mechanisms may participate in this regulation. The aim of this research was to investigate the influence of obesity and metabolic syndrome (MetS) features in clock gene methylation and the involvement of these epigenetic modifications in the outcomes. Sixty normal-weight, overweight and obese women followed a 16-weeks weight reduction program. DNA methylation levels at different CpG sites of CLOCK, BMAL1 and PER2 genes were analyzed by Sequenom's MassARRAY in white blood cells obtained before the treatment. Statistical differences between normal-weight and overweight + obese subjects were found in the methylation status of different CpG sites of CLOCK (CpGs 1, 5-6, 8 and 11-14) and, with lower statistical significance, in BMAL1 (CpGs 6-7, 8, 15 and 16-17). The methylation pattern of different CpG sites of the three genes showed significant associations with anthropometric parameters such as body mass index and adiposity, and with a MetS score. Moreover, the baseline methylation levels of CLOCK CpG 1 and PER2 CpGs 2-3 and 25 correlated with the magnitude of weight loss. Interestingly, the percentage of methylation of CLOCK CpGs 1 and 8 showed associations with the intake of monounsaturated and polyunsaturated fatty acids. This study demonstrates for the first time an association between methylation status of CpG sites located in clock genes (CLOCK, BMAL1 and PER2) with obesity, MetS and weight loss. Moreover, the methylation status of different CpG sites in CLOCK and PER2 could be used as biomarkers of weight-loss success, particularly CLOCK CPGs 5-6.