Tembusu virus (TMUV) infection most commonly affects breeder and layer ducks during laying period, and can also affect young ducks below 7 weeks of age. Here, we report our investigation of a TMUV-caused fatal disease of Jingding ducklings (Anas platyrhynchos domesticus) in Northeast China. The disease resulted in mortalities of up to 40 % in 2 to 4-week-old ducks, up to 25 % in 5 to 6-week-old ducks, and less than 10 % in 7 to 8-week-old ducks. Using a TMUV-specific reverse transcription-PCR assay, all 44 ducks collected from 10 different farms were found positive for TMUV. Phylogenetic analysis of the E nucleotide sequence revealed that five of the six TMUV strains detected from three young ducks and three laying ducks were grouped within cluster 2.1. Inoculation of the liver sample of a 40-day-old sick duck in BHK-21 cells resulted in isolation of cluster 2.1 TMUV strain H. In experimental infections performed using 3-week-old Pekin ducklings (Anas platyrhynchos domesticus) (n = 30; 10 birds/group), high mortality (60 %) was caused by strain H, in sharp contrast with a very low mortality (10 %) caused by strain Y which was isolated during outbreaks of the TMUV-related disease of young Jinding ducks in 2014 in the same region. These findings clearly demonstrated that the cluster 2.1 TMUV strain H is more pathogenic for 3-week-old ducklings as compared to the cluster 2.2 TMUV strain Y. The present study may enhance our understanding of pathogenicity of TMUV in young ducks, and will stimulate further studies on the pathogenesis of TMUV infection.