Hemorrhagic fever with renal syndrome (HFRS) is confirmed by the isolation of hantavirus from serum, detection of virus-specific IgM, or a four-fold change in IgG titers during the acute and convalescent periods measured using an immunofluorescence assay (IFA). However, these tests are inefficient for early diagnosis. Therefore, this study investigated the usefulness of reverse-transcriptase nested polymerase chain reaction (RT-nPCR) for early diagnosis of HFRS using clinical samples such as urine and serum. Electronic medical records of eight patients with confirmed HFRS using IFA and RT-nPCR between May 2016 and May 2020 at Chosun University Hospital were reviewed. The virus was detected in all patients using RT-nPCR targeting the large (L) segment of hantavirus during the early phase in urine and serum. Importantly, the virus was identified in urine at a time when it was not identified in serum. Additionally, the virus was detected in urine and serum for up to 1 month after initial presentation with illness, but not in saliva, using RT-nPCR. We report eight HFRS cases diagnosed using urine and serum, but not using saliva, with RT-nPCR targeting the L-segment. Hantavirus RNA detection by RT-nPCR in urine and serum may aid the rapid diagnosis of HFRS during the early phase of the disease. In particular, HFRS should not be ruled out based on negative RT-PCR results in serum, and RT-PCR should be performed using urine as well as serum during the early phase of symptoms.