Rodent-borne hantaviruses cause two severe acute diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus pulmonary syndrome (HPS; also called hantavirus cardiopulmonary syndrome [HCPS]) in the Americas. Puumala virus (PUUV) is the most common causative agent of HFRS in Europe. Current routine diagnostic methods are based on serological analyses and can yield inconclusive results. Hantavirus-infected patients are viremic during the early phase of disease; therefore, detection of viral RNA genomes can be a valuable complement to existing serological methods. However, the high genomic sequence diversity of PUUV has hampered the development of molecular diagnostics, and currently no real-time reverse transcription-quantitative (RT)-PCR assay is available for routine diagnosis of HFRS. Here, we present a novel PUUV RT-PCR assay. The assay was validated for routine diagnosis of HFRS on samples collected in Sweden during the winter season from 2013 to 2014. The assay allowed detection of PUUV RNA in 98.7% of confirmed clinical HFRS samples collected within 8 days after symptomatic onset. In summary, this study shows that real-time RT-PCR can be a reliable alternative to serological tests during the early phase of HFRS.