Wetland assessment techniques generally have focused on rapid evaluations of local and site impacts; however, wetland biodiversity is often influenced both by adjacent and regional land use. Forty wetlands were studied in the Red River Valley, Southwest Prairie, and the Northern Hardwood Forest ecoregions of Minnesota, USA to assess the strength of association between local and landscape condition and animal community composition. We examined the relationship between the richness of four wetland taxa (amphibians, birds, fish, and invertebrates) and several local and landscape factors (connectedness, wetland isolation, road density, and site disturbances). Landscape variables were calculated for three spatial scales at 500 m (79 ha), 1000m (314 ha), and 2500 m (1963 ha). The four organismal groups studied responded differently across scales, landscape measures, and ecoregions. Bird assemblages were found to have significant relationships (p < 0.05) with percent connectedness across all spatial scales, wetland isolation, and site disturbances in the agricultural Red River Valley and Southwest Prairie ecoregions. In the Southwest Prairie ecoregion, the ratio of bird wetland taxa to upland taxs showed a strong relationship to percent connectedness at the 2500 m scale. Fish assemblages were found to have a significant relationship (p < 0.005) with wetland isolation in the urbanized Hardwood Forest and agricultural Southwest Prairie ecoregion. Few significant relationships were found between amphibian and invertebrate assemblages and landscape variables. A more comprehensive sampling strategy may be required to detect these relationships. Development of effective wetland assessment criteria based on land use-biota relationships should include extensive field community characterization, comprehensive land use analysis, and validation of criteria across wetland types and ecoregions that are specific to an organismal group.