The aim of the present study was to assess the plasma fatty acid composition of the total plasma lipids and lipid fractions in obese prepubertal children with and without metabolic syndrome (MS). Thirty-four obese prepubertal children were recruited: 17 who met MS criteria and 17 who did not; and twenty prepubertal children of normal weight. MS characteristics, insulin resistance (by homeostasis model assessment [HOMA-IR]), and plasma adiponectin (by radioimmunoassay) were recorded. Separation of lipid fractions was performed by liquid chromatography and the concentration of fatty acids in total plasma lipids and fractions was determined by gas-liquid chromatography. Concentrations of 16:1n-7, 16:1n-9, 18:3n-3, 22:6n-3, and n-3 PUFA in total plasma lipids (P < 0.05) and of 16:0, 16:1n-7, 18:1n-9, 18:2n-6, and n-6 PUFA in triacylglycerols (TG) (P < 0.05) were significantly higher in obese MS versus normal-weight children. Increased risk of MS was positively associated with plasma concentration of 16:1n-7 and negatively associated with proportion of 20:4n-6 (OR 2.76; P = 0.004; OR 0.56, P = 0.030, respectively). Saturated FA in TG were associated with HOMA-IR (R = 0.349, P = 0.017) and 22:5n-6 with adiponectin (R = 0.336, P = 0.05). In conclusion, increased concentrations of 16:1n-7 and decreased proportions of 20:4n-6 and 22:5n-6 in plasma lipids appear to be early markers of MS in children at prepubertal age.