Background: Since the turn of the 21 st century, there have been several epidemic outbreaks of poultry diseases caused by Tembusu virus (TMUV). Although multiple mosquito and poultry-derived strains of TMUV have been isolated, no data exist about their comparative study, origin, evolution, and dissemination. Methodology: Parallel virology was used to investigate the phenotypes of duck and mosquito-derived isolates of TMUV. Molecular biology and bioinformatics methods were employed to investigate the genetic characteristics and evolution of TMUV. Principal findings: The plaque diameter of duck-derived isolates of TMUV was larger than that of mosquito-derived isolates. The cytopathic effect (CPE) in mammalian cells occurred more rapidly induced by duck-derived isolates than by mosquito-derived isolates. Furthermore, duck-derived isolates required less time to reach maximum titer, and exhibited higher viral titer. These findings suggested that poultry -derived TMUV isolates were more invasive and had greater expansion capability than the mosquito-derived isolates in mammalian cells. Variations in amino acid loci in TMUV E gene sequence revealed two mutated amino acid loci in strains isolated from Malaysia, Thailand, and Chinese mainland compared with the prototypical strain of the virus (MM1775). Furthermore, TMUV isolates from the Chinese mainland had six common variations in the E gene loci that differed from the Southeast Asian strains. Phylogenetic analysis indicated that TMUV did not exhibit a species barrier in avian species and consisted of two lineages: the Southeast Asian and the Chinese mainland lineages. Molecular traceability studies revealed that the recent common evolutionary ancestor of TMUV might have appeared before 1934 and that Malaysia, Thailand and Shandong Province of China represent the three main sources related to TMUV spread. Conclusions: The current broad distribution of TMUV strains in Southeast Asia and Chinese mainland exhibited longer-range diffusion and larger-scale propagation. Therefore, in addition to China, other Asian and European countries linked to Asia have used improved measures to detect and monitor TMUV related diseases to prevent epidemics in poultry. (C) 2017 Elsevier B.V. All rights reserved.