Hantaviruses are negative-strand RNA viruses that contain three segmented (L/M/S) genome and belong to the genus hantavirus of the family Bunyaviridae. Due to such an unique structure of segmented RNA genome, hantaviruses have a possibility to produce reassortants that containing genomic sets mixed with different segments originated from both parental viruses during the genetic interaction. To investigate whether this phenomenon occurs in vitro, Hantaan (HTN) and Seoul (SEO) viruses were co-infected into Vero-E6 cells and virulent Maaji (MAA) virus was superinfected into avirulent Prospect Hill (PH) virus-infected Vero-E6 cells, respectively. To select only reassortants among progeny viruses, well separated plaque clones were analyzed by multiplex RT-PCR. The putative reassortant viruses detected by 1st multiplex RT-PCR were plaque-purified three times and confirmed by 2nd multiplex RT-PCR. Only 3 reassortants like HTN/HTN/SEO, SEO/HTN/HTN and SEO/HTN/SEO and only 2 reassortants like PH/MAA/MAA, MAA/MAA/PH as designated in order of L/M/S of genomic segments have been identified so far. These results indicate that genetic reassortment can be induced by mixed-infection of two more distantly related serotypes of hantavirus. Interestingly, reassortant SEO/HTN/SEO containing HTN viral M RNA segment is isolated more frequently. This implies that preferential selection of M genome segments occurred when RNA genomes were packaged into virion and also the process of packaging of RNA segments into virion is not random phenomenon. These reassortants would be helpful to know whether genetic reassortment is dependent on genetic distance between hantaviruses and which viral RNA segment plays an important role in coding for virulence marker. Therefore, genetic reassortment can be useful genetic tool to understand genetical, and biological function of hantavirus.