Seroprevalence of Neospora caninum in dairy buffaloes (Bubalus bubalis) was assessed in the Lahore District of Punjab Province, Pakistan. The study revealed an overall prevalence of 54.7% for N. caninum antibodies determined through a competitive enzyme-linked immunosorbent assay performed on randomly collected serum samples. The highest prevalence was observed in buffaloes >3–5 yr of age (64.1%), followed by 57.9% for 5 to 6 yr olds, and 55.8% in 1-yr-old neonates, with high probability of infection under intensive dairy farming conditions. The pattern of prevalence was closely associated with the season as reflected by the highest prevalence (70.5%) in summer (May–August) and lowest (39.6%) in winter (November–January). Aborting buffaloes illustrated significantly higher (78.9%) exposure compared with non-aborting dams (59.8%). Prevalence in animals with canine contact was significantly higher (60.3%) than without contact (48.1%). This is the first reported prevalence of N. caninum in Pakistan.