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Hantaviruses is an emerging infectious disease. Although HCPS has been reported in several regions of Brazil, more cases of HCPS have recently been reported in Minas Gerais than in any other state. In 2009, we analyzed 27 samples presenting antibodies against hantaviruses. These samples originated from 688 symptomatic patients, as determined based on the Hemorrhagic Fever Protocol. A subsequent SYBR Green-based real-time RT-PCR demonstrated the presence of the virus in 22 of the samples. Among the RT-PCR-positive samples, 17 were analyzed using DNA sequencing; these sequences were compared with others deposited in GenBank and showed similarity with the Araraquara and Juquitiba virus clusters. This work describe the detection of Juquitiba virus, including three fatal cases, in Minas Gerais state, furthermore, showed that it is feasible to characterize the circulating strains using a small fragment of S segment. Finally, the results suggest the co-circulation of Araraquara and Juquitiba virus in a single biome in Minas Gerais state. J. Med. Virol. 87:725–732, 2015. © 2015 Wiley Periodicals, Inc.