This report describes our diagnostic findings on isolation and characterization of a Tambusu virus (TMUV) infection in a broiler breeder flock, which caused a sharp decline in egg production and primary clinical symptoms of lethargy, reduced appetite and watery diarrhea. The main autopsy lesions observed in affected breeder chickens were reproductive system abnormalities, including follicular membrane hemorrhage and follicular liquefaction. This TMUV infection resulted in about 1.8% mortality in eight weeks. The TMUV isolation was made from oviduct specimens and conducted in both embryonated chicken eggs and chicken embryo fibroblast cells cultures. Amino acid analysis of the virus structure of envelope protein (E protein) indicated that the TMUV isolate belonged to the same branch of duck TMUV vaccine strains of FX2010-180P, DF2 and HB, which were widely used in domestic ducks in China. Notably, the chicken TMUV isolate exhibited the highest homology (99.0%) with the TMUV DF2 and HB vaccine strains. The isolation and characterization of chicken-derived TMUV in this study brings an urgent need for further investigations into the impact of TMUV infections on egg-type chickens to enhance prevention and control strategies for better performance of egglaying hens of commercial layer farms and boiler breeder flocks.