Neospora caninum has been implicated as a sporadic cause of abortion and perinatal deaths in sheep flocks globally. However, its significance as a reproductive pathogen for sheep in Australia remains unknown. The aims of this study were to (i) determine the seroprevalence of N. caninum in Australian breeding ewes and (ii) examine if natural exposure to N. caninum is associated with poor reproductive performance of primiparous ewes in southern Australia. Thirty flocks of primiparous ewes (aged 1-2years old at lambing) from 28 farms in three states (Western Australia, South Australia and Victoria) were monitored between mating and lamb marking. Blood samples were also collected from multiparous mature ewes (aged 3years or older) at each farm. Seroprevalence for anti-N. caninum IgG using indirect ELISA was determined for a subset of primiparous ewes that were predominantly determined to be pregnant and subsequently failed to rear a lamb (n = 1279) and randomly selected mature multiparous ewes with unknown reproductive status (n = 558). Neopsora caninum apparent seroprevalence was 0.16% (95% confidence interval 0.03%, 0.5%) in primiparous ewes, with seropositivity identified in two ewes from farms located in South Australia and Victoria. There was no evidence of seropositivity in mature ewes with apparent seroprevalence 0% (0%, 0.45%). These findings suggest that N. caninum infection was not widespread in primiparous ewes or mature multiparous ewes on these farms, and exposure to N. caninum infection was unlikely to explain abortion and perinatal mortalities observed for primiparous ewes.