Gallibacterium anatis biovar haemolytica has been suggested to have a causal role in peritonitis and salpingitis in chickens. Therefore, the aim of this study was to investigate the occurrence of G. anatis biovar haemolytica in chickens with reproductive disorders. One hundred and forty one birds from 31 layer flocks were submitted for necropsy and the following organs were examined for bacteria: choana, trachea, lung, heart, liver, spleen, ovary, oviduct, duodenum and cloaca. Examination for Escherichia coli was included as it can induce reproductive disorders. G. anatis was isolated in pure culture from the reproductive tract of affected birds in six of the 31 flocks while E. coli was obtained in pure culture from 10 of them. Both G. anatis and E. coli were isolated from the reproductive tract of 14 of the 31 flocks. The genetic diversity of the Gallibacterium isolates was assessed by amplified fragment length polymorphism on a subset of 83 isolates. Generally, each flock was infected with a single clone, which could be isolated from various sites in the birds. However, in two flocks, the majority of birds yielded positive samples from the internal organs, indicating that these particular clones may be more invasive. The findings support previous suggestions that G. anatis biovar haemolytica is associated with infection of the reproductive tract of chickens, making it a likely cause of lowered productivity and an animal welfare concern.