The oomycete Pythium comprises one of the most important groups of seedling pathogens affecting soybean. There has been limited research on Pythium spp. pathogenic on soybean in the northern Great Plains. The objectives of this research were to isolate and identify Pythium spp. infecting soybean in North Dakota and to test their pathogenicity. Identification of Pythium spp. was achieved using molecular techniques and morphological features. A total of 26 known Pythium spp. and three unknown species were recovered from soybean seedling roots collected from 125 fields between 2011 and 2012. In 2011, the three most abundant species isolated were P. ultimum, Pythium sp. (unknown; GenBank HQ643777.1), and P. heterothallicum, representing 21, 16, and 12% of 2,675 isolates, respectively. More species and isolates were obtained in 2011, a wet and cool year, compared with 2012, which was dry and warm. The majority of Pythium spp. caused pre-emergence damping-off on soybean with less than 50% emergence in a 2-week test using infested soil at 23°C. In contrast, in the presence of P. orthogonon, P. nunn, or P. rostratifingens there was approximately 80% or greater emergence and most plants survived for several weeks, although lesions were observed on roots. Mortierella spp., a zygomycete, was commonly isolated along with Pythium spp. in 2012, but not in 2011. This is the first report of P. kashmirense, P. minus, P. periilum, P. rostratifingens, P. terrestris, P. viniferum, and P. violae as pathogens of soybean seedlings. In addition, this is the first report of P. kashmirense, P. viniferum, and P. terrestris in the United States.