During the initiation of neonatal pulmonary respiration, there is an exponential increase in reactive oxygen species that must be scavenged by antioxidant defences. However, neonate and preterm newborns are known to possess immature antioxidant mechanisms to neutralize these toxic effects. The purposes of this study were to compare the development of antioxidant system between preterm and term canine neonates and to evaluate the magnitude of acid–base balance during the initial 4 h of life. A prospective study was conducted involving 18 neonatal puppies assigned to Term Group (63 days of gestation; n = 5), Preterm‐57 Group (57 days of gestation; n = 8) and Preterm‐55 Group (55 days of gestation; n = 5). Neonates were physically examined through Apgar score and venous haemogasometry within 5 min, 2 and 4 h after birth. No difference on amniotic fluid and serum superoxide dismutase (SOD), glutathione peroxidase (GPx) and the marker of oxidative stress (thiobarbituric acid reactive substances; TBARS) was verified. Irrespective of prematurity, all neonates presented low vitality, hypothermia, acidosis, hypoxaemia and hypercapnia at birth. However, term puppies clinically evolved more rapidly than preterm newborns. During the course of the study, premature neonates presented more severe complications, such as prolonged hypoxaemia and even death. In conclusion, premature puppies have no signs of immature enzymatic mechanisms for controlling oxidative stress, although SOD and GPx may participate in achieving acid–base balance. Aside from initial unremarkable symptoms, premature puppies should be carefully followed up, as they are at high risk of succumbing to odds of prematurity.