Japanese encephalitis virus (JEV) is a flavivirus that is maintained via transmission between Culex spp. mosquitoes and water birds across a large swath of southern Asia and northern Australia. Currently JEV is the leading cause of vaccine-preventable encephalitis in humans in Asia. Five genotypes of JEV (G-I–G-V) have been responsible for historical and current outbreaks in endemic regions, and G-I and G-III co-circulate throughout Southern Asia. While G-III has historically been the dominant genotype worldwide, G-I has gradually but steadily displaced G-III. The objective of this study was to better understand the phenomenon of genotype displacement for JEV by evaluating both avian host and mosquito vector susceptibilities to infection with representatives from both G-I and G-III. Since ducks and Culex quinquefasciatus mosquitoes are prevalent avian hosts and vectors perpetuating JEV transmission in JE endemic areas, experimental evaluation of virus replication in these species was considered to approximate the natural conditions necessary for studying the role of host, vectors and viral fitness in the JEV genotype displacement context. We evaluated viremia in ducklings infected with G-I and G-III, and did not detect differences in magnitude or duration of viremia. Testing the same viruses in mosquitoes revealed that the rates of infection, dissemination and transmission were higher in virus strains belonging to G-I than G-III, and that the extrinsic incubation period was shorter for the G-I strains. These data suggest that the characteristics of JEV infection of mosquitoes but not of ducklings, may have play a role in genotype displacement.