Colibacillosis associated with salpingitis occurred in a layer chicken flock on a commercial egg-producing farm in Japan. An increase in mortality was observed when the birds were at 62 weeks of age and reached 0.89% at 68 weeks of age. Postmortem examinations revealed pericarditis, perihepatitis, airsacculitis, and reproductive tract lesions in 4 affected birds at 69 weeks of age. Analysis of pulse-field gel electrophoresis (PFGE) patterns and putative virulence genes of 22 E. coli isolates obtained from the affected birds demonstrated that isolates from liver, heart, and the surface of the reproductive tract of one bird were genetically unrelated with those recovered from the lumen of the oviduct. In the other birds, isolates from liver, heart, and reproductive tract lesions were closely related to each other. These findings suggest that salpingitis in the former bird may be caused by ascending infection of the oviduct from the cloaca and salpingitis in the remaining birds may occur as part of systemic infection.