Hantaviruses possess three RNA segments of negative sense. Co-infection of closely related hantaviruses may result in generation of a progeny virus with genomic polyploidy, containing a partial or complete set of genome originated from more than one parental virus. To characterize the formation of viral genomic polyploidy, cultured Vero-E6 cells were co-infected with two closely related hantaviruses, Hantaan and Maaji, and the progeny viruses examined. The genotype of plaque-purified viruses was analyzed by a virus-specific RT-PCR. Seventy percent (67/96) of the progeny virus was categorized as Hantaan and 3.3% (2/96) was classified as Maaji, whereas 20% (21/96) was considered polyploidy as they contained both types of the S RNA segment. Most of the polyploidy progeny viruses were unstable and gave rise to either one of the parental viruses or a reassortant after several rounds of plaque purification. No recombination between the heterologous pair of S RNA was observed for those polyploid viruses during three consecutive plaque-to-plaque passages. These data suggest that the viral polyploidy formation constitutes a primary mechanism underlying the generation of a newly emerged hantavirus.