Background Pulse oximetry is a vital tool for screening critical congenital heart disease (CCHD) in newborns. However, fixed thresholds may not account for physiological variations caused by altitude. This study evaluates the impact of elevation on normal neonatal oxygen saturation (SpO₂) in Jordan, spanning sites from moderate altitude to below sea level. Methods In a cross-sectional study, 149 healthy, full-term newborns were screened at three hospitals located at 1,050 m (Amman), sea level (Aqaba), and 420 m below sea level (Dead Sea). Preductal and postductal SpO₂ measurements were taken between 24 and 72 hours of life using standardized protocols. Statistical analyses, including analysis of variance (ANOVA) and Tukey's honestly significant difference (HSD), were used to compare SpO₂ levels across altitudes. Results Mean upper-limb SpO₂ was significantly lower at 1,050 m (96.6%) compared to sea level (98.0%) and sub-sea level (97.8%) (p < 0.001). Similar trends were observed for lower-limb SpO₂. No significant differences were found between sea-level and sub-sea-level sites. Despite lower SpO₂ at moderate altitude, no newborns met the American Academy of Pediatrics (AAP) criteria for CCHD. Conclusion Moderate altitude results in a statistically significant, but modest, reduction in neonatal SpO₂, potentially increasing false-positive rates in CCHD screening. Our findings support the development of altitude-specific screening protocols to improve diagnostic accuracy and reduce unnecessary interventions. Further research with larger and more diverse populations is needed to inform altitude-adjusted guidelines, particularly in geographically varied regions like Jordan.