Highly pathogenic avian influenza has not been reported in Nepal to date. Surveillance for the presence of avian influenza viruses was conducted in 16 districts of Nepal from February 2004 to December 2005. Four hundred forty-six serum samples were collected from ducks, chickens, and pigeons and tested for antibodies to all influenza A viruses by competitive enzyme-linked immunosorbent assay (C-ELISA). Any sera positive by C-ELISA were tested for antibodies to H5, H7, and H9 influenza viruses by hemagglutination inhibition tests. One hundred and thirty-five cloacal swabs from healthy ducks and chickens were tested by commercial avian influenza antigen detection kits. A further 13 tissue samples from diseased birds were tested for the presence of virus by virus isolation in eggs, cell culture, and immunohistochemistry. No influenza viruses were detected in any of the tissues or swabs. All serum samples collected before October 2005 were negative for antibodies. The first sera positive for antibodies were collected on October 13, 2005, which were determined to be of the H9N2 subtype. This is the first report of serologic evidence of an avian influenza virus infection in Nepal.