Glucose, lactate and cortisol concentrations in amniotic fluid were measured at birth in 95 pups and related to neonatal viability based on Apgar scoring and to neonatal mortality. Neither amniotic parameters nor neonatal mortality were associated with the Apgar score. Stillborn pups showed high lactate (P < 0.001) and cortisol (P < 0.05) but low glucose amniotic concentrations (P < 0.001). No amniotic fluid differences were observed between normal and malformed pups. Amniotic glucose (P < 0.001), lactate (P < 0.05) and cortisol (P < 0.05) concentrations were higher in pups delivered by vaginal parturition than by Caesarean section. Birth weight was higher in live pups than in pups dying within 48 h (P < 0.05). Although these are preliminary results, the analysis of amniotic fluid collected at birth could be a valuable predictor of neonatal outcomes in dogs.