Objective: To investigate the relationship between physical fitness index (PFI) and body mass index (BMI) of Tibetan children and adolescents in different high-altitude areas in Tibet, China. Methods: Using the stratified cluster sampling method, 3819 Tibetan children and adolescents from three different high-altitude areas including Nyingchi, Lhasa and Nagqu in the Tibet area of China were given grip strength, standing long jump, sitting forward bend, 50 m running and endurance running tests. One-way analysis of variance was used to compare the physical fitness index in different high-altitude areas. In addition, the method of curve regression analysis was used to analyze the relationship between PFI and BMI. Results: In general, the level of PFI in Nagqu, Tibet, China was lower than that in Nyingchi and Lhasa, and the levels of girls were generally lower than those of boys. The proportions of malnourished, normal, overweight and obese Tibetan boys in high-altitude areas were 11.8%, 79.7%, and 8.5%; those of girls were 3.3%, 82.3%, and 14.4%, respectively. The curve regression analysis showed that the model fitting of male Nyingchi, Lhasa, Nagqu and female Nyingchi, Lhasa, Nagqu were all significant (F values were 29.697, 34.709, 37.500, 9.123, 9.785, 6.939, p < 0.01). The relationship between BMI and PFI generally showed an inverted “U” curve relationship. Conclusion: The negative impact of overweight and obesity on physical fitness of Tibetan boys in high-altitude areas is significantly higher than that of girls, and the negative impact of overweight and obesity on physical fitness of boys in Lhasa and Nyingchi area is more significant than that in the Nagqu area. In the future, attention should be paid to Lhasa and the occurrence of overweight and obesity among Tibetan boys in Nyingchi area in order to prevent the sharp decline of physical fitness and promote the physical and mental development of Tibetan children and adolescents in high-altitude areas.