Although the phenomenon of innate resistance to flaviviruses in mice was recognized many years ago, it was only recently that the genetic locus (Fiv) controlling this resistance was mapped to mouse Chromosome (Chr) 5. Here we report the fine mapping of the Flv locus, using 12 microsatellite markers which have recently been developed for mouse Chr 5. The new markers were genotyped in 325 backcross mice of both (C3H/HeJ x C3H/RV)F-1 x C3H/HeJ and (BALB/c x C3H/RV)F-1 x BALB/c backgrounds, relative to Fly. The composite genetic map that has been constructed identifies three novel microsatellite loci, D5Mit68, D5Mit159, and D5Mit242, tightly linked to the Fly locus. One of those loci, D5Mit159, showed no recombinations with Fly in any of the backcross mice analyzed, indicating tight linkage (43.3 cM). The other two, D5Mit68 and D5Mit242, exhibited two and one recombinations with Fly (0.6 and 0.3 cM) respectively, defining the proximal and distal boundaries of a 0.9-cM segment around this locus. The proximal flanking marker, D5Mit68, maps to a segment on mouse Chr 5 homologous to human Chr 4. This, together with the previous data produced by our group, locates Fly to a region on mouse Chr 5 carrying segments that are conserved on either human Chr 4, 12, or 7, but present knowledge does not allow precise identification of the syntenic element.