Salmonella is one of the most common causes of food-borne outbreaks and infection worldwide. The gold standard detection method of Salmonella is cultivation. With time-consuming cultivation, there is a need to investigate rapid and accurate processes. The study evaluated different approaches to detect Salmonella in poultry feces samples. Poultry farm feces samples from 21 cities in Iran were collected from January 2016 to December 2019. Microbiological cultures, serological assays, and multiplex PCR (m-PCR) were used to detect and characterize Salmonella spp. isolates. Serological assays and m-PCR were used to determine the serogroups A, B, C1, C2, D1, E, H, and FliC. The m-PCR was used for the detection of seven Salmonella serovars and a chi-square test was performed to compare the discriminatory power of the methods. Out of 2300 poultry feces samples, 173 (7.5%) and 166 (7.2%) samples were detected as Salmonella spp. by cultivation and m-PCR, respectively. The sensitivity of the molecular method was equal to cultivation at 0.96 (CI = 95%).Assessment of H antigenic subgroups showed the same for both m-PCR and serological tests. Therefore, the matching rate of the two methods for detection of all H antigenic subgroups was 100%. Thus, the relationship between the results obtained from both methods was signi cant in the contingency table test (P < 0.01). The PCR-based approach con rmed the detection of Salmonella in a shorter period (24-36 hours) compared to the conventional microbiological approach (3-8 days).