A cross-sectional survey was conducted in Sudan to determine sero-prevalence and risk factors associated with Neospora caninum infection in nonvaccinated dairy herds and to assess importance of the disease. Blood samples were collected from a total of 262 animals from 25 herds. Sera were tested for antibodies against N. caninum using ELISA test. The prevalence rates of N. caninum antibodies in cattle were high both at herd level (44%) and at individual animal level (10.7%). Herd level infection rates were similar in Khartoum State (43.7%) and at Gazira States (44.4%). The overall prevalence rates were higher (16.1%) in Gazira State than in Khartoum State (9%) but with no significant variation. The sero-prevalence at individual animal level was significantly higher (p<0.05) in animals with history of abortion (12.8%) than in apparently healthy animal (11.3%), animal with history of infertility (8.1%), or neonatal death of calves (4.3%). In addition, significantly higher (P<0.05) sero-prevalence was observed in samples collected during the rainy season (6.87%) than winter (3.05%) or summer (0.76%). However, no significant differences in sero-prevalence due to locality, animal breed, sex, and age were observed (p > 0.05). This preliminary study reveals for the first time the existence of natural N. caninum infection in Sudan. Also, the findings of the present study indicated that this disease is highly prevalent in two major areas of dairy production in the country, and this calls for control strategy to be implemented.