This study investigated differences in health-related fitness (20-m shuttle run, handgrip, bent arm hang, standing long jump, shuttle run 4 x 10 m and sit and reach tests) in 2474 Spanish adolescents (1196 boys and 1278 girls; age 13-18.5 years) classed as underweight, normal weight, overweight or obese according to body mass index. Body fat and fat-free mass were derived from skinfold thickness. The prevalence of underweight was higher than obesity in girls (4.8% vs 3.0%, respectively; P < 0.05) and the opposite in boys (3.9% vs 5.8%, respectively; P < 0.05). Underweight was associated with a higher performance in the bent arm hang test in girls (P < 0.05) and a lower performance in handgrip in both genders (P < 0.01) compared with normal weight. Overweight and obese adolescents presented a lower performance in 20-m shuttle run, bent arm hang, standing long jump and shuttle run 4 x 10 m tests (P < 0.001), but a higher performance in handgrip strength (P < 0.001) compared with normal weight. In weight-bearing tests, the association became non-significant after adjusting for fat mass. In conclusion, not only overweight and obesity but also underweight seem to be determinants of health-related fitness in adolescents. The associations could be related to differences in body composition.