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Tembusu virus (TMUV), a significant member of the Orthoflavivirus genus, poses a severe threat to poultry, particularly ducks, causing several neurological disorders and decreased egg production. This study examined the virological characteristics of TMUV isolated from ducks in southern Vietnam, a region with limited prior research on this pathogen. We performed a biopsy and analyzed six brain and liver samples from ducks exhibiting symptoms indicative of TMUV infection. We used real-time polymerase chain reaction, isolated the virus from the samples using a cell culture approach with C6/36 and BHK-21 cell lines, and characterized the viral sequences using nextgeneration sequencing methods. The virus was present in a majority of the samples. TMUVs replicated and formed cytopathic effects and plaques in C6/36 and BHK-21 cells. The virus was successfully isolated from five samples, and its complete genome sequences for these five strains were obtained and deposited into GenBank. There was a significant genomic similarity to TMUV strains found in China, with more than 90% genetic concordance. In conclusion, our TMUV isolates are crucial for understanding the virus's distribution and guiding the development of practical diagnostic tools, vaccines, and control strategies in the poultry industry. A broader geographical spread of TMUVs and potential cross-border epidemiological links warrant further research. [ABSTRACT FROM AUTHOR]