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The soundscape composition of freshwater habitats is poorly understood. Our goal was to document the occurrence of biological sounds in a large variety of freshwater habitats over a large geographic area. The underwater soundscape was sampled in freshwater habitat categorized as brook/creek, pond/lake, or river, from five major river systems in North America (Connecticut, Kennebec, Merrimack, Presumpscot, and Saco) over a five-week period in the spring of 2008. Over 7,000 sounds were measured from 2,750 minutes of recording in 173 locations, and classified into major anthropophony (airplane, boat, traffic, train and other noise) and biophony (fish air movement, also known as air passage, other fish, insect-like, bird, and other biological) sound categories. Anthropogenic noise dominated the soundscape of all habitats averaging 15 % of time per recording compared to less than 2 % for biological sounds. Anthropophony occurred in 79 % of recordings and was mainly due to traffic and boat sounds, which exhibited significant differences among habitats and between non-tidal and tidal river regions. Most biophonic sounds were from unidentified insect-like, air movement fish, and other fish sound sources that occurred in 57 % of recordings. Mean frequencies of anthropogenic noises overlapped strongly with the biophony, and comparisons of spectra suggest that insect- like and air movement sounds may be more susceptible to masking than other fish sounds. There was a significant decline in biodiversity and biophony with increasing ambient sound levels. Our poor understanding of the biophony of freshwater ecosystems, together with an apparent high temporal exposure to anthropogenic noise across all habitats, suggest a critical need for studies aimed at identification of biophonic sound sources and assessment of potential threats from anthropogenic noises.