Cases of mpox have been reported in several European countries, including Spain. Our objective was to evaluate the usefulness of serum and nasopharyngeal samples for diagnosis of mpox. The presence of MPXV DNA was studied using real-time PCR (CerTestBiotec, Zaragoza, Spain) in 106 samples from 50 patients: 32 skin, 31 anogenital, 25 sera, and 18 nasopharyngeal/pharyngeal, in the Hospital Clínico Universitario of Zaragoza (Spain). Sixty-three samples from twenty-seven patients were MPXV PCR-positive. The real-time PCR Ct values in the anogenital and skin samples were lower than serum and nasopharyngeal samples. More than 90% of anogenital (95.7%), serum (94.4%), and skin (92.9%) samples were real-time PCR-positive. Eighteen (66.7%) of the twenty-seven patients who were MPXV PCR-positive had antecedents or presented with one to three sexually transmitted infection (STI) agents. Our results indicate that the use of serum samples can help facilitate the diagnosis of MPXV infections.