[b]Introduction and objective[/b]. Tick-borne infections constitute an increasing health problem in dogs and may lead to death, especially in young or elderly individuals. Canine babesiosis constitutes a serious health problem in dogs worldwide. The aim of the study was to verify the probability of vertical transmission of [i]Babesia canis[/i] between the bitch and the pups. [b]Materials and methods[/b]. In Autumn 2011, cases of babesiosis were diagnosed in a litter of 6-week-old puppies of a Central Asian Shepherd dog. Immediately following the first case of infection, blood samples were collected from all the pups in the litter (n=10) and from the female. Detection of Babesia infection was performed by molecular and microscopical techniques. [b]Results.[/b] The presence of[i] B. canis [/i]DNA was detected using PCR in three pups, presenting at the time or 24–48 hours later with babesiosis symptoms, and in their asymptomatic mother. The isolates derived from the pups and the female – 520 bp 18S rRNA gene fragment – were compared and analyzed. All isolates from the pups and their mother were identical and showed 100% homology with [i]B. canis[/i] group B (EU622793), supporting the same source of infection. Additionally, the USG of the peritoneal cavity was performed in the female, presenting evidence for splenomegaly. [b]Conclusions.[/b] On the basis of (1) the same timing of three pup cases; (2) the identical [i]B. canis[/i] sequences derived from all positive dogs; (3) evident splenomegaly in the asymptomatic female, this provides the first evidence of the vertical transmission of this piroplasm in dogs.