Objective: To examine the multiplication efficiency Japanese encephalitis virus (JEV) genotype Ⅰ (GⅠ) and genotype Ⅲ (GⅢ) of different cell lines which originated from human, porcine, mosquitoes in order to prove mechanism of JEV GⅠ replacement JEV GⅢ since it emerging in nature recent decades. Methods: The mixture of GI and GIII JEV isolates was inoculated on human rhabdomyosarcoma (RD), pig kidney epithelial (PS) and Aedes albopictus C6/36 clone (C6/36) which originated from human, porcine and mosquitoes, respectively. Plaque assays were performed to calculate virus titer and real-time RT-PCR with GI and GIII specific primer sets to quantify the number of GI and GIII RNA copies. Results: The highest virus titer reached at the 3rd day of post infection when GⅠ and GⅢ mixture was inoculated on RD and PS and that of C6/36 was at the 4th day. JEVs were amplified and maintained by C6/36 cells after 10 passages whereas that by RD and PS only limited within 8 and 6 passages, respectively. GⅠstrain amplified and maintained more efficiently on C6/36 and PS but not RD, whereas GⅢ strain amplified and maintained more efficiently on RD. Conclusions:There is a correlation between the multiplication efficiency of GⅠand GⅢ JEV strains when these two genotype strains co-infected on different cell lines with the predominance of GⅠstrains in C6/36 and PS and the limited detection of GⅠ strains in RD cells proving a possible mechanism of shift JEV genotypes in nature recent decades since GⅠ emerging.