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Introduction: Low oxygen saturation suggests severe heart or respiratory disease. Most practitioners consider less than 95% as abnormal oxygen saturation in infants. Experts have recommended this cut-off value based on large-sample studies at or near sea level. However, there are few large-sample studies of infants born at low to moderate altitudes. The aim of this study is to determine threshold values of newborn oxygen saturation at low to moderate altitudes. Methods: We included 35 hospitals located in the Yunnan province of China, with altitudes varying from 267 to 2,202 meters. The hospitals recruited 41,097 consecutively born, asymptomatic neonates between Aug. 2015 and Jun. 2016. Obstetric nurses measured pre- and post-ductal oxygen saturation of each baby at 24 hours of age and before hospital discharge. Results: We categorized all births, according to the altitude of birth, into three groups: low altitude (0 to 500 meters), low to moderate altitude (500 to 1,500 meters), and moderate altitude (1,500 to 2,500 meters). The mean of pre-ductal oxygen saturation measured at 24 hours of age was 97.9% at low attitudes, 96.4% at low to moderate altitudes and 95.5% at moderate attitudes. We considered the 2.5th percentile as the cut off threshold, and recommended cut-off values of 95% for low altitudes, 93% for low to moderate altitudes, and 92% for moderate altitudes. Conclusion: These findings provide revised threshold values of oxygen saturation for pediatricians and obstetricians when screening newborns for severe congenital heart and lung disease at low to moderate altitudes. [ABSTRACT FROM AUTHOR]