Salmonella Enteritidis (SE) is frequently isolated from poultry and humans. Chicken meat and egg are two important sources of SE infection for humans. This study was conducted to detect the SE infection in Iranian poultry farms using serological methods. A number of 8208 serum samples were provided from 171 poultry flocks (pullet, commercial layer, broiler breeder, layer breeder, grandparent breeder, and broiler) and were analyzed by rapid slide agglutination test (RSA) and ELISA. All samples were negative in RSA but ~ 45%l of samples (from 112 flocks) contained anti-SE antibody in ELISA. The titres ranged 0.476-0.590, which were at low level and showed the value of ELISA vs. RSA. The results indicated that the poultry flocks had been infected with SE at least once during the course of production. This is the first comprehensive study, at this sample size, in Iran regarding the serologic profile of SE in poultry flocks and its findings are very important for poultry industry and the control strategy for SE infection.