Hantaviruses are negative-strand RNA viruses that contain three segmented genome and belong to the family Bunyaviridae. Due to such an unique structure of segmented RNA genome, these viruses have a possibility to produce reassortant that have genomic sets mixed with different segments originated from both parental virus during the genetic interaction. To investigate whether this phenomenon occurs actually, Hantaan virus (HTN) could be coinfected with Seoul virus (SEO) to Vero-E6 cell. And also, HTN could be coinfected with Prospect hill virus (PH) to investigate the rate of genetic reassortment according to the genetic distance among the HTN, SEO and PH viruses. As a available method to differentiate the reassortant, we used the multiplex RT-PCR that detect and differentiate the serotype of hantaviruses in mixed infection. Each progeny clone could be screened by multiplex RT-PCR. So, we have constructed the multiplex RT-PCR system that separated amplifications for each segment of Hantaviruses. Our multiplex RT-PCR system provide sensitive, specific and simplified tools for the rapid differentiation of hantaviruses serotypes and also the diagnosis of Hantavirus infections.