A strain of Zika virus (P6-740) was isolated from one of 58 pools of 1,277 Aedes aegypti mosquitoes collected in cities and towns of peninsular Malaya. The mosquitoes in the positive pool were collected from shop houses in Bentong, a small town in West Central Malaya. No strains of Zika virus were isolated from 59 pools of 4,492 Aedes albopictus collected in suburban and rural areas and in rubber plantations, nor from any of 179 pools of 27,636 mosquitoes of 23 other Aedes species collected in rural areas, rain forests, mangrove swamps, and fresh-water swamp forests throughout Malaya. P6-740 was readily identified as a strain of Zika virus by the use of antiserum from monkeys in the standard hemagglutination-inhibition test and the plaque-reduction neutralization test in Vero cell cultures. Specific identification of P6-740 as Zika virus was also made in cross-neutralization tests in mice with hyperimmune mouse serum prepared to itself, Zika, Spondweni, and other group B arboviruses. The latter method, however, was less specific than the plaque-reduction neutralization test for comparison of Zika and Spondweni viruses.