BACKGROUND Wild aquatic birds, reservoir of low-pathogenicity (LP) avian influenza viruses (AIVs), congregate in huge numbers in Western Siberia wetlands, where major intra- and inter-continental bird flyways overlap. In 2005 and 2006, highly pathogenic (HP) AIV H5N1 epizootics affected wild and domestic birds in the Novosibirsk Region. In 2012, we evaluated AIV persistence in Siberian natural and anthropic ecosystems. METHODOLOGY/PRINCIPAL FINDINGS In Novosibirsk Region, 166 wild birds ecologically linked to aquatic environments and 152 domestic waterfowl were examined for AIV isolation in embryonating chicken eggs. Biological samples were obtained by integrating the conventional cloacal swab collection with the harvesting of samples from birds' plumage. Haemagglutinating allantoic fluids were further characterized by serological and molecular methods. In August-September 2012, 17 AIVs, including three H3N8, eight H4N6, two H4N?, one H2N?, one H?N2, and two unsubtyped LPAIVs, were isolated from 15 wild ducks. Whereas comparable proportions of wild Anseriformes (n.118) tested virus isolation (VI)-positive from cloaca and feathers (5.9% vs 8.5%) were detected, the overall prevalence of virus isolation, obtained from both sampling methods, was 2.4 times higher than that calculated on results from cloacal swab examination only (14.4% vs 5.9%). Unlike previously described in this area, the H4N6 antigenic subtype was found to be the prevalent one in 2012. Both cloacal and feather samples collected from domestic waterfowl tested VI-negative. CONCLUSION/SIGNIFICANCE We found lack of evidence for the H5N1 HPAIV circulation, explainable by the poor environmental fitness of HPAIVs in natural ecosystems. Our LPAIV isolation data emphasise the importance of Siberia wetlands in influenza A virus ecology, providing evidence of changes in circulation dynamics of HN antigenic subtypes harboured in wild bird reservoirs. Further studies of isolates, based on bioinformatic approaches to virus molecular evolution and phylogenesis, will be needed to better elucidate mechanisms involved in AIV perpetuation in this area.