Zika virus (ZIKV) is a flavivirus transmitted by the Aedes spp mosquito. Originally discovered in Uganda, Africa, ZIKV has then emerged in the Pacific Islands, and more recently the Americas. In 2015, ZIKV was responsible for a major outbreak, which began in Brazil and spread across both continents of the Americas, resulting in more than 700,000 autochthonous cases with over 3500 cases of congenital Zika syndrome. Here we employed our recently developed bacterium-free method to generate directly from a published sequence the outbreak- and microcephaly-associated Natal 2015 isolate, the non-outbreak associated Malaysian 1966 P6-740 isolate, as well as chimeric viruses with the structural prM-E genes of Natal 2015 isolate replaced with those from Malaysian 1966 P6-740 or African 1947 MR766 isolates. By performing multistep growth kinetics on mouse embryonic fibroblasts (MEF), we have found that MR766 and P6-740 replicated more efficiently than Natal, which is likely due to the repeated passages that MR766 and P6-740 isolates historically undergone through in suckling mice. Similarly, we showed that Natal infection was asymptomatic in IFNAR-/- mice, while MR766 produced high viraemia and caused significant mortality in these mice. Comparing replication efficiencies in MEF of chimeric Natal viruses containing prM-E genes of MR766 and P6-740 with the parental isolates showed that the increased mouse cell tropism is encoded primarily within the prM-E structural proteins of MR766 isolate, although additional determinants of mouse cell tropism were also encoded in the non-structural proteins region. Our findings caution the use of archival ZIKV isolates and call for more critical interpretation of the data obtained in the mouse models of infection using these isolates.