Avian Influenza Virus (AIV)-H9 subtype was reported to be endemic in Asia and Middle East. It induces considerable economic losses in poultry industry and was involved in human infection. In the present study, attempts were made to estimate the RT-PCR-based prevalence of Avian Influenza Virus-H9N2 subtype in the Eastern Region of Saudi Arabia during the period from January 2012 to March 2014. Tissue samples were collected from 115 flocks of broiler chicken from targeted region during the study period. Part of the Hemagglutinin (HA) gene was directly sequenced to determine circulating genotype. Samples from four flocks were positive to AIV-H9 subtype. Sequencing and phylogenetic analysis of the four detections showed high nucleotide identity to each other and to previous AIV-H9N2 isolates from Saudi Arabia, UAE and Israel. The four detections belong to G1 lineage of the H9N2 subtype. AIV-H9 subtype seems to be of low prevalence in broiler chicken of the Eastern region of Saudi Arabia. Further studies to determine biologic and pathologic characterizations of these detections and to determine prevalence of other AIV subtypes in Saudi Arabia are required to build control and prevention strategy and to minimize the threat it pose to public health.