Avian influenza virus (AIV) has been recognized as one of the most important pathogens in poultry. This study was designed to investigate the prevalence of AIV H9 subtype in commercial chicken flocks in Jordan by serological and molecular methods. Serum samples from 180 chicken flocks (120 broilers and 60 layers) free from respiratory symptoms, were examined by hemagglutination inhibition (HI) test for specific antibodies against AIV H9 subtype, and 83 chicken flocks (60 broilers and 23 layers) with respiratory symptoms, were examined by reverse transcription-polymerase chain reaction (RT-PCR) using universal primers for influenza A viruses, then specific primers targeting AIV H9 gene were used for the flocks that were positive by universal primers. Overall, 65 out of 120 broiler flocks (54.2%), and 47 out of 60 layer flocks (78.3%) were positive for AIV H9 subtype antibodies. Nucleic acid of influenza A viruses was detected in 31 out of 60 broiler flocks (51.7%), and 15 out of 23 layer flocks (65.2%). AIV H9 subtype was detected in all flocks that were positive for influenza A viruses. The current study confirmed the endemic nature of AIV H9 subtype in broiler and layer flocks in Jordan. It is essential that the biosecurity on poultry farms should be improved to prevent the introduction and dissemination of influenza and other viruses. Furthermore, farmers need to be educated about the signs, lesions, and the importance of this virus.