Streptococcus gallolyticus subsp. gallolyticus causes endocarditis in humans and acute septicemia in domestic birds. We describe here the infective endocarditis caused by the bacterium found among clinically healthy broilers at two abattoirs in Japan. The chickens were thought to be healthy because of the lack of clinical symptoms and normal levels of mortality before slaughtering. At the time of inspection, some chickens were condemned because of organ disorders characterized by vegetative valvular endocarditis as well as focal necrosis in heart, liver, and spleen. Streptococcus gallolyticus subsp. gallolyticus was isolated from the organs as a pure culture, indicating that the bacterium probably was the causative agent of the disorders. Amplified fragment length polymorphism analysis of the isolates collected at the abattoirs from chickens grown in nine different farms indicated that the isolates were different variants of the same clonal lineage and may have been derived from the same ancestor. These results suggest that S. gallolyticus subsp. gallolyticus causes infectious endocarditis in chickens and that healthy chickens may possess the bacterium in their normal flora as an opportunistic pathogen.