Toxoplasma gondii is an emerging risk to marine mammals globally and is known to be present in New Zealand waters. Archived Sera of New Zealand sea lions (Phocarctos hookeri) from two recolonizing mainland populations at the Otago Peninsula and Stewart Island as well as a declining population at Enderby Island in the New Zealand sub-Antarctic were tested for antibodies to T.gondii. Sera were screened using commercially available ELISA and latex agglutination tests (LAT) and then confirmed with western blot analysis. Antibodies were found in 13.6% (3/22) of mainland samples but not sub-Antarctic samples. The positive LAT titres in three adult females were strong (1:2048-4096) and in two of these animals persistent for up to two years, but there was no evidence of associated clinical signs or reproductive failure. Continuing surveillance is pertinent to assess subclinical and clinical impacts of T.gondii on these threatened populations.