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Hantavirus Pulmonary Syndrome is an, often fatal, emerging zoonotic disease in the Americas caused by hantaviruses (family: Hantaviridae). In Brazil, hantavirus routine diagnosis is based on serology (IgM-ELISA) while RT-PCR is often used to confirm acute infection. A Semi-nested RT-PCR and an internally controlled RT-qPCR assays were developed for detection and quantification of four hantaviruses strains circulating in the Brazilian Amazon: Anajatuba (ANAJV) and Castelo dos Sonhos (CASV) strains of Andes virus (ANDV) species; and Rio Mamoré (RIOMV) and Laguna Negra (LNV) strains of LNV species. A consensus region in the N gene of these hantaviruses was used to design the primer sets and a hydrolysis probe. In vitro transcribed RNA was diluted in standards with known concentration. MS2 bacteriophage RNA was detected together with hantavirus RNA as an exogenous control in a duplex reaction. RT-qPCR efficiency was around 100% and the limit of detection was 0.9 copies/μL of RNA for RT-qPCR and 10 copies/μL of RNA for Semi-nested RT-PCR. There was no amplification of either negative samples or samples positive to other pathogens. To assess the protocol for clinical sensitivity, specificity and general accuracy values, both assays were used to test two groups of samples: one comprising patients with disease (n = 50) and other containing samples from healthy individuals (n = 50), according to IgM-ELISA results. A third group of samples (n = 27) infected with other pathogens were tested for specificity analysis. RT-qPCR was more sensitive than semi-nested RT-PCR, being able to detect three samples undetected by conventional RT-PCR. RT-qPCR clinical sensitivity, specificity and general accuracy values were 92.5%, 100% and 97.63%, respectively. Thus, the assays developed in this study were able to detect the four Brazilian Amazon hantaviruses with good specificity and sensitivity, and may become powerful tools in diagnostic, surveillance and research applications of these and possibly other hantaviruses. Author summary: Hantavirus Pulmonary Syndrome (HPS) is a serious and often fatal disease caused by viruses known as hantaviruses. These viruses are harbored by wild rodents and people can become infected through contact with infected-rodents droppings, urine or saliva. After an incubation time of 1–8 weeks, patients usually present flu-like symptoms such as fever, fatigue and muscle aches, although some patients may also present headaches, dizziness, chills, nausea, vomiting, diarrhea, and abdominal pain. It is only 4–10 days after initial symptoms, however, that the severe stage of disease takes place. Symptoms include coughing, shortness of breath and eventually the lungs fill with fluid which can lead to shock and death. As such, HPS should be diagnosed quickly as any delay may have great impact on patient recovery. However, given the unspecific nature of early symptoms, clinical diagnosis of HPS is difficult and laboratory assays are needed to confirm hantavirus infection as soon as possible, helping physicians to choose the most adequate treatment. In this study, we developed new laboratory assays that can help detect the virus in infected patients in early stages of disease. In addition, we showed these assays have a good performance in discriminating HPS from other similar diseases by testing not only several samples collected from both HPS patients and healthy individuals but also samples infected with other pathogens. Our results show that these assays may become important tools for rapid, sensitive and specific diagnosis of HPS. [ABSTRACT FROM AUTHOR]