Background The causative agent of plague, Yersinia pestis , is maintained in nature via a flea-rodent cycle. Western Iran is an old focus for plague, and recent data indicate that rodents and dogs in this region have serological evidence of Y . pestis infection. The purpose of this study was to conduct a large-scale investigation of Y . pestis infection in shepherd dogs, rodents, and their fleas in old foci for plague in Western Iran. Materials and methods This study was conducted in Hamadan province from 2014 to 2020. Rodents and fleas were collected from various locations throughout this region. Y . pestis was investigated in rodent spleen samples and fleas using culture, serology, and real-time PCR methods. Additionally, sera samples were collected from carnivores and hares in this region, and the IgG antibody against the Y . pestis F1 antigen was assessed using an ELISA. Results In this study, 927 rodents were captured, with Meriones spp. (91.8%) and Microtus qazvinensis (2.6%) being the most prevalent. A total of 6051 fleas were collected from rodents and carnivores, most of which were isolated from Meriones persicus . None of the rodents or fleas examined tested positive for Y . pestis using real-time PCR and culture methods. Meanwhile, IgG antibodies were detected in 0.32% of rodents. All serologically positive rodents belonged to M . persicus . Furthermore, none of the sera from the 138 carnivores (129 sheepdogs, five Vulpes vulpes , four Canis aureus ), and nine hares tested positive in the ELISA test. Conclusion This primary survey of rodent reservoirs shows serological evidence of Y . pestis infection. Western Iran is an endemic plague focus, and as such, it requires ongoing surveillance.