The aim of this study was to determine the seroprevalence of avian influenzaH9N2 subtype in the industrial ostrich farms and its geographical distribution. This cross-sectional study was conducted from January to June 2015. A total of 40 farms were selected from different provinces of Iran, from each of which 11 ostriches (n=440) were sampled. The sera samples were examined using 4 hemagglutination units of H9N2 antigens. A frequency distribution was used to describe the responses to the survey questions. The mean titers between provinces were compared using one-way analysis of variance. According to the results, 21 (47.5%) out of 40 farms and 108 (24.5%) out of 440 ostriches tested positive in the HI-H9N2 test. There were statistically significant differences between the mean titers of samples in different provinces (P<0.001). The current study was conducted on unvaccinated ostriches. The results showed that H9N2 had a high seroprevalence at both farm and bird levels. The findings of this study can be for the further investigation of infection in ostrich farms in order to consider this species in the surveillance programs of the Iranian Veterinary Organization. The detection and isolation of viruses and epidemiological investigation are necessary for the persistent use of H9N2 vaccines in some ostrich farms.