Twenty-one dairy herds with a history of Neospora caninum-associated abortions were used for a longitudinal serological study. A total of 1676 animals were blood sampled 3 times and used to evaluate a single serological screening for N. caninum antibodies. The results of the first serological screening were compared with the results based on three consecutive samples, whereby two or more positive or negative test results per animal were considered to determine its serological status as positive or negative, respectively. In both test regimes 95.3% of the animals had the same interpretation, of which 33.9% were seropositive, and 61.3% seronegative. Relative sensitivity of one-time sampling compared to three consecutive samplings was 94.7%, while relative specificity was 95.6%. Relative specificity differed between herds. Predictive values positive and negative of one-time sampling were 92.4 and 97%, respectively. The agreement between one-time sampling and three consecutive samplings, kappa, was 0.90. For evaluation of discrepant results age distribution and pedigree data were used to provide clues regarding likelihood of transmission. Age clustering of seropositive animals was interpreted to indicate a point source infection. Daughter-mother relationships were used for the interpretation of congenital infections. The proportion of congenital infection decreased with increasing parity of the mother. Seropositive heifers had 80% congenitally infected offspring, while in older cows 66% of the offspring was congenitally infected, possibly due an increased immunity to transplacental infection with age. It is concluded that a single serological screening of a whole herd in connection with an analysis of age distribution and pedigree data is a rapid and valid method to interpret the serologic status of individual animals and to study the mode of transmission of N. caninum.