Lake Ontario and St. Lawrence River (LOSL) wetland bird abundance and diversity are greatly influenced by lake and river hydrology. Our study used an interdisciplinary ecosystem approach, blending avian and plant ecology, ecohydraulic, statistical ecology and modelling to evaluate potential impacts of water level fluctuations on indicator species representative of the wetland breeding bird assemblages in the entire LOSL freshwater system. Multi-year (2000-2003) bird surveys captured bird distribution and density in wetland habitats under varying degrees of water inandation, depth and fluctuation. Analyses revealed strong associations between estimated breeding pair densities and plant communities, water depth, and degree of water level fluctuation during the breeding season for a suite of wetland bird species using marsh, wet meadow, shrub swamp and treed swamp habitats. These quantitative associations were used to develop wetland bird performance indicators for use in a LOSL water regulation review study. Several bird species also nest at or near the water surface and are thus vulnerable to nest flooding or stranding. Changes to the seasonal hydrology of Lake Ontario and St. Lawrence River that result in an increased frequency or magnitude of these nest failure events may have a significant impact on regional population sustainability. Long term nest record databases were analyzed to create nesting flooding and stranding probability equations based on water level increases and decreases during the breeding season. These species-specific nesting relationships were incorporated into a reproduction index. Many breeding bird species were strongly associated with specific wetland plant communities. Predicted habitat suitability, as measured by estimated breeding pair density, can also change significantly within a specific wetland plant community based solely on changes in water depth during the breeding season. Three indicator species, Black Tern, Least Bittern and Virginia Rail were selected as key environmental performance indicators for alternate regulation plan comparisons. Water regulation criteria should be such that the long term diversity and abundance of wetland plant communities and frequency of spring flooding in marsh habitats during breeding are not reduced. Magnitude and frequency of water level change during the nesting season (May-July) can also adversely impact reproductive success of many wetland bird species. As such, regulation criteria that increase the seasonal magnitude and frequency of water level change may be detrimental to the long term viability of certain regional breeding bird populations.