Active and passive smoking in pregnant women is associated with perinatal morbidity and mortality risk, including abortion, preterm birth, low birthweight, and malformations. No data are available on intrauterine exposure to smoking during pregnancy in dogs. This study aimed to fill this gap by exploring the detectability and quantity of cotinine, the major metabolite of nicotine, in maternal (serum and hair) and newborn (amniotic fluid and hair) biospecimens collected at birth in dogs. For this purpose, twelve pregnant bitches, six exposed to the owner’s smoke and six unexposed, were enrolled. A further six non-pregnant bitches exposed to passive smoke were included to investigate the role of pregnancy status on cotinine uptake. Exposed dogs, dams, and puppies had greater cotinine concentrations than unexposed ones. Although without statistical significance, serum and hair cotinine concentrations were higher in pregnant compared to non-pregnant bitches, suggesting a different sensitivity to tobacco smoke exposure during gestation. The present results provide evidence for cotinine transplacental passage in the dog. It is conceivable that fragile patients such as pregnant, lactating, and neonate dogs may be more susceptible to the harmful effects of second-hand smoke exposure. Owners should be sensitized to the risk of smoke exposure for their pets.