The clinical presentation of Mpox (monkeypox) resembles that of several other sexually transmitted infections. Although cases of Mpox are thought to be overlooked in the clinical setting, no study has investigated whether this is the case in Japan. In this study, we retrospectively screened suspected cases of syphilis to investigate overlooked cases of Mpox in Japan. We retrospectively analyzed 88 saliva samples and 78 whole blood samples from 88 patients treated in general internal medicine and infectious disease clinics. Molecular diagnostic testing revealed Mpox virus DNA in the saliva and blood samples of 1 patient with suspected primary syphilis in April 2023. This case was retrospectively diagnosed as Mpox. The findings of this study indicate that health care providers should be aware of Mpox as a differential diagnosis in patients with sexually transmitted infections. Improved access to molecular diagnostic testing may reduce the risk of overlooked cases of Mpox. Retrospective screening using saliva samples could help to increase opportunities to intervene against Mpox transmission.