Introduction. Among the protozoa of veterinary importance, Neospora caninum is responsible for large economic and productive losses in cattle herds. Dogs are being considered the definitive hosts of the parasite. Neospora caninum has gained considerable attention through its role in the etiology of bovine abortion. The current study aimed at assessing the status of Neospora infection in cattle in Boyer-Ahmad County in Kohgiluyeh and Boyer-Ahmad province, southwest of Iran. Methods. In this cross-sectional study, 150 cattle blood samples were collected and samples were screened for N. caninum antibodies using a modified direct agglutination test (MAT). For the same samples, 130 buffy coats were collected and tested, by PCR, for the presence of N. caninum DNA, targeting the Nc-5 gene. Results. Anti-N. caninum antibodies were detected in the sera of 49 out of 150 cattle which is corresponding to a seroprevalence rate of 32.7%. Neospora DNA was detected in buffy coats of 26 out of 127 (20.47%) cattle. Even though Neospora infection was more common in females and in five-year-old cattle by serology and molecular methods, yet there was no statistically significant difference between age, sex, and Neospora infection in both molecular and serological methods (
p
>
0.05
). Conclusion. Findings of the current study indicate a high rate of N. caninum infection in cattle of Boyer-Ahmad region in the southwest of Iran. This issue should be further investigated and the prevention and control of this parasite in livestock, due to the high financial burden of this parasite in the livestock industry, should be considered.