Pathogenesis and reservoir host adaptation of animal and zoonotic viruses are poorly understood due to missing adequate cell culture and animal models. The bank vole (Myodes glareolus) and common vole (Microtus arvalis) serve as hosts for a variety of zoonotic pathogens. For a better understanding of virus association to a putative animal host, we generated two novel cell lines from bank voles of different evolutionary lineages and two common vole cell lines and assayed their susceptibility, replication and cytopathogenic effect (CPE) formation for rodent-borne, suspected to be rodent-associated or viruses with no obvious rodent association. Already established bank vole cell line BVK168, used as control, was susceptible to almost all viruses tested and efficiently produced infectious virus for almost all of them. The Puumala orthohantavirus strain Vranica/Hällnäs showed efficient replication in a new bank vole kidney cell line, but not in the other four bank and common vole cell lines. Tula orthohantavirus replicated in the kidney cell line of common voles, but was hampered in its replication in the other cell lines. Several zoonotic viruses, such as Cowpox virus, Vaccinia virus, Rift Valley fever virus, and Encephalomyocarditis virus 1 replicated in all cell lines with CPE formation. West Nile virus, Usutu virus, Sindbis virus and Tick-borne encephalitis virus replicated only in a part of the cell lines, perhaps indicating cell line specific factors involved in replication. Rodent specific viruses differed in their replication potential: Murine gammaherpesvirus-68 replicated in the four tested vole cell lines, whereas murine norovirus failed to infect almost all cell lines. Schmallenberg virus and Foot-and-mouth disease virus replicated in some of the cell lines, although these viruses have never been associated to rodents. In conclusion, these newly developed cell lines may represent useful tools to study virus-cell interactions and to identify and characterize host cell factors involved in replication of rodent associated viruses.