The Valentine Formation of A. L. Lugn, which is of late Miocene and early Pliocene age, contains diatomaceous and plant-bearing sediments at a locality on the north bluff of the Niobrara River, about 10 miles south of Kilgore, Cherry County, Nebr. The fossil flora has been studied by H. D. MacGinitie, who concluded that the deposit is of late Miocene age. Correlative strata near Valentine, Nebr., several miles east of this locality, are reported to contain a vertebrate fauna of late Miocene age. The Kilgore diatom assemblage consists of 45 taxa of nonmarine diatoms. Of these, four species are herein recorded as new, one species has been previously reported from the middle Pliocene of France, and the remaining 40 taxa are identified as still living in modern nonmarine environments. The diatom assemblage suggests deposition in a relatively shallow, small lake. The waters of this lake were probably hard, and perhaps at times somewhat alkaline or saline. It is difficult to separate the influences of ecology and geologic age on the composition of the diatom assemblage, but this problem should be resolved as other Tertiary assemblages are studied. The five presumably extinct species may prove to be useful for stratigraphic correlation when their geologic ranges are better known. The composition of the assemblage, the relative number of taxa, and the absence of several widespread modern genera and species of nonmarine diatoms should be of value in identifying other late Miocene assemblages and in distinguishing them from assemblages of different age.