Nine isolates of a Colletotrichum sp. collected in North America on yellow water-lily (Nuphar luteum subsp. polysepalum) at five locations in the Pacific Northwest (PNW) and one isolate from Nymphaea odorata in Rhode Island were compared to three isolates of C. nymphaeae occurring on Nymphaea alba and Nuphar luteum in Europe. The appressoria of all North American isolates were significantly wider than those of C. nymphaeae. Conidia of the nine PNW isolates were significantly wider than those of both the Rhode Island isolate and C. nymphaeae isolates. All isolates were compared using random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism of the ITS region (RFLP-ITS). Two distinct clusters were differentiated in the dendrogram based on the RAPD analysis. The ten North American isolates formed one cluster and the European isolates of C. nymphaeae formed a second cluster. The separation of the North American isolates from the European isolates was supported by distinct restriction digest phenotypes of the ITS region. Based on morphological and molecular characterization the North American fungus is proposed as a new species, C. nupharicola.