The type and the amount of dietary fat have a significant influence on the metabolic pathways involved in the development of obesity, metabolic syndrome, diabetes type 2 and cardiovascular diseases. However, it is unknown to what extent this modulation is achieved through DNA methylation. We assessed the effects of cholesterol intake, the proportion of energy intake derived from fat, the ratio of polyunsaturated fatty acids (PUFA) to saturated fatty acids (SFA), the ratio of monounsaturated fatty acids (MUFA) to SFA, and the ratio of MUFA+PUFA to SFA on genome-wide DNA methylation patterns in normal-weight and obese children. We determined the genome-wide methylation profile in the blood of 69 Greek preadolescents (∼10 years old) as well as their dietary intake for two consecutive weekdays and one weekend day. The methylation levels of one CpG island shore and four sites were significantly correlated with total fat intake. The methylation levels of 2 islands, 11 island shores and 16 sites were significantly correlated with PUFA/SFA; of 9 islands, 26 island shores and 158 sites with MUFA/SFA; and of 10 islands, 40 island shores and 130 sites with (MUFA+PUFA)/SFA. We found significant gene enrichment in 34 pathways for PUFA/SFA, including the leptin pathway, and a significant enrichment in 5 pathways for (MUFA+PUFA)/SFA. Our results suggest that specific changes in DNA methylation may have an important role in the mechanisms involved in the physiological responses to different types of dietary fat.