Leaves of wheat infected with the leaf rust fungus Puccinia triticina were obtained from farm fields and breeding plots at experimental stations in the Great Plains, Ohio River Valley, and southeastern states in 2016 in order to identify virulence phenotypes prevalent in the United States in different wheat-growing regions. In total, 496 single uredinial isolates derived from the leaf rust collections were tested for virulence to 20 lines of Thatcher wheat that differ for single leaf rust resistance genes. In total, 71 virulence phenotypes were described in the United States in 2016. The three most common virulence phenotypes across the United States were MBTNB, MBDSD, and TNBJJ. Phenotype MBTNB is virulent to Lr11, and was most common in the soft red winter wheat region of the southeastern states and Ohio Valley. Phenotype MBDSD is virulent to Lr17 and Lr39, and was most common in the hard red winter wheat area of the southern Great Plains. Phenotype TNBJJ is virulent to Lr24 and Lr39, which are present in the hard red winter wheat cultivars. The P. triticina population in the United States was characterized by two major regional groups of virulence phenotypes in the Great Plains region where hard red winter and spring wheat cultivars are grown, and in the southeastern states and Ohio Valley region where soft red winter wheat cultivars are grown. Isolates from New York State differed the most for virulence compared with the other two major regions.