Children aged 11–15 years from three low-fluoride zones (< 0.5 ppm in drinking water) situated at sea level, 1,500 m and 2,400 m above sea level, and from two higher-fluoride zones (0.5–1.0 ppm in drinking water) were examined for dental fluorosis. In the low-F zones 36.4% of the children at sea level had dental fluorosis, as compared to 78.0% at 1,500 m, and 100.0% at 2,400 m. In the higher-F zones 71.2% had dental fluorosis at sea levels as compared to 93.8% at 1,500 m (p < 0.001). The intraoral pattern of dental fluorosis was similar in each of these five populations, and the severity of dental fluorosis of each tooth type increased significantly with increases in altitude in both the low and higher-fluoride zones (p < 0.001). Moreover the severity of dental fluorosis was associated with increases in fluoride concentrations at constant altitudes (at sea level and at 1,500 m). This study establishes for the first time that populations living at high altitudes may be more susceptible to dental fluorosis than those at low altitudes for a given concentration of fluoride in drinking water. The findings indicate that certain physiological changes may occur in humans living at high altitude whereby the effects of fluoride on mineralizing tissues become exacerbated.