Ornithobacteriosis is an infectious disease of avian species that has been reported in almost all countries around the world, except Thailand. The objectives of this study were to determine the seroprevalence of Ornithobacterium rhinotracheale (ORT) and to isolate and identify ORT in broilers and broiler breeders in Thailand. Chicken antibodies had been randomly checked from 17 farms (19 flocks) of broilers and 23 farms (28 flocks) of broiler breeders. The seropositive flocks were 63% and 100% in broilers and broiler breeders, respectively. The sera analysis showed that the individual 280 broiler sera antibody responses were 67.5% negative, 12.9% suspect, and 19.6% positive. The individual antibody responses of 510 broiler breeder sera revealed 12.2% negative, 38.0% suspect, and 49.8% positive samples. The bacteria were isolated and identified by polymerase chain reaction (PCR). Bacterial isolation and identification revealed that nine isolates of the 12 PCR analysis samples showed positive results to PCR analysis. All the positive PCR samples were collected from the broiler breeder farms.