The epidemiological information has obtained on avian influenza virus (AIV) in eastern Hokkaido, Japan, where AIV surveillance has not been performed. Cloacal or fecal samples obtained from migratory water birds were screened for AIV both by real-time reverse transcriptase polymerase chain reaction to detect the influenza A virus matrix (M) gene and by egg inoculation. Between 2007 and 2009, a total of 2,488 samples were collected from various avian species in Abashiri, Kushiro, Nemuro and Tokachi districts of eastern Hokkaido. AIVs were isolated from 18 of those samples (0.7%). No AIV was isolated from the 1,449 samples collected in Abashiri, Kushiro and Nemuro districts, although 6 were positive for the M gene by RRT-PCR. In contrast, 52 (5.0%) of the 1,039 samples collected from ducks in Tokachi district were M gene positive; AIVs were isolated from 18 of those samples (1.7%). The isolates included H3N5 (1 isolate), H3N6 (1), H3N8 (9), H4N2 (1), H4N6 (2), H6N5 (1), H6N8 (1), and H11N3 (2) subtypes. H3N5 and H11N3 subtypes have not been frequently isolated, and our study is the first to report H3N5 and the second to report H11N3 in Japan. Phylogenetic analysis revealed that the M genes of all isolates belonged to the Eurasian lineage.