In recent years, the H5N1 subtype of avian influenza virus (AIV) has become an important zoonotic pathogen. The surveillance of AIV in its natural host, the waterfowl, is crucial to monitoring and controlling the disease in poultry and other species. In this study, we report on the isolation of H5 AIV from cloacal swabs of waterfowl captured in Minnesota and South Dakota. We screened a total of 7260 cloacal samples from waterfowl using matrix gene–directed, real-time reverse transcription–(rRT-PCR) and H5-specific rRT-PCR and found 148 samples to be positive for the H5 subtype. On inoculation of 71 of these samples in embryonated chicken eggs, 25 samples yielded H5 AIV. On subtyping with N-specific primers, we detected a mixture of subtypes in 15 isolates. Molecular pathotyping confirmed the isolated H5 subtypes to be low pathogenicity avian influenza. Continuation of AIV surveillance programs should help in understanding the epidemiology and ecology of AIV.