The sudden emergence of Ornithobacterium rhinotracheale (ORT) in commercially raised poultry species and its presence in non-galliform birds raise important epidemiological issues about the role of interspecies transmission. In the present study, 21 ORT strains isolated from pigeons and from birds of prey were analyzed using the recently established multilocus sequence typing (MLST) scheme. Results were compared to MLST sequence data available from ORT strains isolated mainly from turkeys and chickens, but also single strains from pheasant, guineafowl and rook. The pigeon-derived ORT strains (n=11) were closely related amongst themselves representing their own cluster distant from ORT strains of non-columbiform avian hosts. ORT strains isolated from birds of prey (n=10) revealed a higher genetic heterogeneity that corresponded well to their host family relationships but grouped within the two mainly poultry- based clusters. None of these strains had a sequence type identical to strains investigated previously. However, three strains isolated from common kestrels and a single strain from a turkey vulture shared one or two out of seven gene loci, respectively, with strains of turkey and chicken origin. The MLST results of ORT isolated from pigeons and birds of prey likely reflect evolutionary bacterial host adaptations but might also indicate a potential for interspecies transmission. Definite conclusions should be drawn carefully as so far a few strains from non-galliform birds were analyzed by MLST. By extending the number of ORT isolates and the range of potential avian hosts, the MLST database can provide a valuable resource in understanding transmission dynamics.