Tissue damage resulting from oxidative stress induced by a pathological condition might have more serious consequences in children than in adults. Researchers have not yet identified particular markers - alone or in combination with others - of oxidative stress, or their role in pediatric diseases. The aim of this study was to identify gender-based biomarkers for measuring oxidative stress. Oxidative biomarkers were studied in 138 healthy Spanish children (85 boys, 53 girls) 7 to 12 years of age, at the prepubertal (Tanner I) stage, independent of body mass index (BMI), age, fitness (measured by 20-m shuttle run test), and physical activity (measured by participation in an after-school exercise program). The oxidative biomarkers measured were lipid peroxidation products, total nitrites, protein carbonyls, and oxidized glutathione (GSSG). The antioxidant biomarkers measured were total glutathione (TG), reduced glutathione (GSH), superoxide dismutase activity (SOD), and glutathione peroxidase activity. In the study population, height, weight, waist circumference, and BMI were lower in girls than in boys. For oxidative biomarkers, boys had higher levels of protein carbonyl than girls (p < 0.001). In spite of this, girls had higher levels of GSSG (p < 0.001) and TG (p = 0.001), and a lower GSH/GSSG ratio (p < 0.001) than boys. For the antioxidant response, girls had higher levels of SOD (p = 0.002) than boys. All analyses were adjusted for BMI, age, fitness, and physical activity. In conclusion, prepubertal girls had higher oxidative stress than boys, in addition to higher levels of SOD, independent of age, BMI, fitness, and physical activity.