A comparative study examining replication and disease pathogenesis associated with low-pathogenic H5N1, H5N2, or H5N3 avian influenza virus (AIV) infection of chickens and ducks was performed. The replication and pathogenesis of highly pathogenic AIV (HPAIV) has received substantial attention; however, the behavior of low-pathogenic AIVs, which serve as precursors to HPAIVs, has received less attention. Thus, chickens or ducks were inoculated with an isolate from a wild bird [A/Mute Swan/MI/451072/06 (H5N1)] or isolates from chickens [A/Ck/PA/13609/93 (H5N2), A/Ck/TX/167280-4/02 (H5N3)], and virus replication, induction of a serological response, and disease pathogenesis were investigated, and the hemagglutinin and neuraminidase (NA) gene sequences of the isolates were determined. Virus isolated from tracheal and cloacal swabs showed that H5N1 replicated better in ducks, whereas H5N2 and H5N3 replicated better in chickens. Comparison of the NA gene sequences showed that chicken-adapted H5N2 and H5N3 isolates both have a deletion of 20 amino acids in the NA stalk region, which was absent in the H5N1 isolate. Histopathological examination of numerous organs showed that H5N2 and H5N3 isolates caused lesions in chickens in a variety of organs, but to a greater extent in the respiratory and intestinal tracts, whereas H5N1 lesions in ducks were observed mainly in the respiratory tract. This study suggests that the H5N1, H5N2, and H5N3 infections occurred at distinct sites in chicken and ducks, and that comparative studies in different model species are needed to better understand the factors influencing the evolution of these viruses.