A longitudinal study was performed to investigate the variations of Neospora caninum antibody levels in individual milk during lactation as well as the association between antibody levels in serum and milk. Serum and milk samples of 15 milking cows were collected between February 2003 and September 2004 in three smallholder dairy farms in Khon Kaen province in northeast Thailand. All samples were analyzed for presence of antibodies by an N. caninum iscom ELISA test kit and the results were given as percent positivity (PP). The effects of time between calving and sampling, lactation number, and season on milk and serum PP were studied using Generalized Estimation Equations methods. All cows were antibody positive in either milk or serum at the first two consecutive samplings. Although serum and milk PP varied considerably, milk PP was consistently positive throughout the study. Cows of all lactation groups had a higher adjusted mean of milk PP at calving compared to later months after calving although the only significant difference was in first lactation. Serum and milk PP were always lower in first lactation than in second and later lactations. An adjusted mean of milk PP for cows classified as having serum PP> or =55 was significantly (P<0.05) higher than that of cows classified as having lower serum PP. Our results indicate that individual milk can be an alternative material to demonstrate presence of N. caninum antibodies in lactating cows.