BACKGROUND: This study was conducted to characterize Newcastle disease virus (NDV) isolates obtained from waterfowl from the Upper Midwest region of the United States. A total of 43 NDVs were isolated by inoculation of cloacal samples in embryonated chicken eggs. These isolates were obtained from 24 mallards, seven American green-winged teals, six northern pintails, four blue-winged teals, and two wood ducks. Partial sequences of fusion gene were analyzed to determine the pathotypes and genotypes involved. RESULTS: Deduced amino acid sequence of the cleavage site of fusion (F) protein revealed that all isolates had avirulent motifs. Of the 43 isolates, 23 exhibited sequence motif of (111)GGKQGRL(117) at the cleavage site, 19 exhibited (111)GEKQGRL(117) while one isolate showed (111)GERQGRL(117). Phylogenetic analysis based on comparison with different classes of NDVs revealed that all 43 isolates clustered with class II NDVs and none with class I NDVs. Within class II, five isolates were phylogenetically close to genotype I NDVs while the remaining 38 were close to genotype II. CONCLUSION: We conclude that more than one genotype of NDV circulates in waterfowl in the Upper Midwest region of the US. Continuous surveillance may help better understand the epidemiology of NDVs maintained in wild bird populations and their relationship to NDVs in domestic poultry, if any.