Although migratory waterfowl is well known to be a major reservoir for avian influenza virus (AIV), there are only few recently published reports about the seroprevalence of AIV in this group of birds. To investigate the AIV antibody status in migratory waterfowl of Iran, we collected 217 serum samples from 25 different species of waterfowl during 2003 and 2004. These serum samples were tested by a competitive ELISA. 77 samples from 14 different species were positive (35.5%). Interestingly, the seroprevalence of antibodies against type A influenza viruses was significantly higher in Anseriformes (64%) than in Non-Anseriformes (12%) and in total birds (35.5%). Our results show furthermore that mallards which winter in Iran in large numbers and show 87.5% positive reactions might play an important role in the epidemiology of influenza virus in this region.