Orthohantaviruses infect humans via inhalation of the viral particles in the excreta of infected rodents or direct contact with infected rodents. The infections caused by Puumala orthohantavirus (PUUV) and Dobrava-Belgrade orthohantavirus (DOBV) have been reported in Turkey. Serum samples of 346 healthy volunteers who are in the high-risk group of Orthohantavirus infections among the residents of Cal, Baklan, Civril, and Bekilli counties, located in the northeast part of Denizli province, were used in this study. The samples were screened and confirmed using commercial ELISA and immunoblot tests, which detect IgG antibodies against DOBV, PUUV, and Hantaan orthohantavirus. IgG antibodies against PUUV were detected in the samples of 2 volunteers (2/346, 0.6%). One was a veterinarian and the other a farmer and they live in the Baklan and Cal counties, respectively, Both of them have a high probability of exposure to the virus, based on their occupation and living conditions. However, no symptoms were found in the clinical findings of both cases. This study is the first publication of reported PUUV seropositivities from the southwestern part of Turkey.