Introduction: Awareness of the species of Shigella has particular importance in the way of dealing with an outbreak, controlling it, and treating patients. The purpose of this study was to use the Multiplex PCR method and comparison with the culture method to detect Shigella species, as well as analysing the antibiotic resistance pattern in its different species. Methods: Simultaneously, the fecal samples of the patients with diarrhea and a sample from each patient in Cary-Blair medium were sent to the laboratory. Cultivation, serotyping, and antibiogram analysis were performed using the samples inoculated in the Cary-Blair medium. DNA extraction of the fecal samples and amplifying of invC, rfc, wbgZ, and rfpB genes were performed. Results: Totally, 300 samples from the patients were analyzed, out of which 240 Shigella isolated (80%) were detected using the culture method and serotyping, and 260 Shigella isolated (86.6%) using the PCR method (Shigella flexneri (77%), Shigella sonnei (19.2%), and Shigella dysenteriae (3.8%)). 20 samples suspicious of Shigella were observed in the culture method but were not identified by serotyping. However, in the PCR assay, they were identified as S. flexneri (n=16) and S. sonnei (n=4). The resistance of Shigella isolated to Co-trimoxazole was observed to be (78.3%), Ceftriaxone (42.5%), Cefixime (42%), Azithromycin (40.7%), Ofloxacin (34.5%), Nalidixic acid (25 %), and Ciprofloxacin (16%). Conclusion: Due to annual outbreaks of various Shigella species in the country, it is recommended that the Multiplex PCR method, be used along with culture method in laboratories to identify Shigella isolated. The antibiogram results showed increasing resistance of Shigella to the available antibiotics.