Background and Objectives: Exclusive breastfeeding is recommended for most newborns. However, exclusively breastfed newborns sometimes experience excess weight loss (EWL, loss ≥10% of birth weight) while lactation is being established. Our primary objective was to evaluate the sensitivity and specificity of the Newborn Weight Loss Tool (NEWT) in early identification of exclusively breastfed newborns who develop EWL; and secondarily, identify breastfeeding variables associated with an at-risk NEWT trajectory. Materials and Methods: We conducted a secondary analysis of prospective data from mother-infant dyads screened for inclusion in the U.S. site of the WHO Growth Reference Study. We excluded records if: NEWT-specific criteria not met, missing key data, or >60 mL formula consumed. We defined NEWT "test-positive" based on an in-hospital weight at about 24 hours falling within the NEWT trajectory consistent with eventual EWL. We defined cases as true EWL based on weight measured at home on day of life 4 (DoL4). Results: Of 280 original records, 60 were excluded (n = 27, NEWT-specific exclusion; n = 15, missing data; n = 18, >60 mL formula), resulting in 220 paired newborn weights measured in-hospital (17 ± 8 hours), and at DoL4 (84 ± 8 hours). NEWT status correctly identified 6/28 EWL cases (21% sensitivity [95% confidence interval, CI, 8-34%]), and 158/192 noncases (82% specificity [95% CI, 75-89%]). NEWT test-positive status was associated with greater weight loss, lower perceived breastfeeding support, and infant less often showing feeding cues on DoL4 (p < 0.05). Conclusion: Sensitivity in predicting EWL is low when applying NEWT at about 24 hours of life; however, early test-positive status is associated with indicators of breastfeeding difficulties on DoL4.