Ornithobacterium rhinotracheale (ORT) is a bacterium common to commercial poultry and wild birds throughout the world. It is also known as a causative agent of respiratory diseases. A total of 93 ORT isolates originating from chickens, pigeons, ostriches, quail, turkeys, and an Asian crested goshawk (Accipiter trivirgatus) in Taiwan, between 2004 and 2006, were used in this study. High genetic similarity (97%–100%) in 16S rRNA sequence was revealed among the 50 randomly selected isolates, in addition to a reference strain (ATCC-51464) and seven reference sequences from GenBank. In order to obtain a greater genetic discrimination among the ORT isolates, random amplified polymorphic DNA (RAPD) and single-enzyme amplified fragment length polymorphism (SE-AFLP) methods were further conducted. The results showed that both RAPD and SE-AFLP assays showed higher discriminatory abilities than the 16S rRNA sequence assay. Genetic clustering revealed that chicken- and quail-origin isolates were genetically distinct from those of the ostrich, pigeon, and Asian crested goshawk-origin isolates. However, among the two typing methods, the turkey-origin isolates showed diverse genetic characteristics to domestic avian species. With this information, ecologic and epidemiologic studies could be furthered for the reduction and control of ORT transmission in Taiwan.