Leaf rust, caused by Puccinia triticina Eriks., is the most common disease of wheat (Triticum aestivum L.) in the United States and worldwide. The objective of this study was to characterize seedling and adult plant leaf rust resistance in hard red spring wheat cultivars grown in Minnesota, North Dakota, and South Dakota, and postulate the identity of the seedling leaf rust resistance genes in the cultivars. Twenty-six cultivars, near-isogenic lines of Thatcher wheat that differ for single leaf rust resistance genes, and three wheat cultivars with known leaf rust resistance genes, were tested with 11 different isolates of leaf rust collected from the United States and Canada. The leaf rust infection types produced on seedling plants of the cultivars in greenhouse tests were compared with the infection types produced by the same isolates on the Thatcher near-isogenic lines to postulate which seedling leaf rust resistance genes were present. Seedling leaf rust resistance genes Lr1, Lr2a, Lr10, Lr16, Lr21, and Lr24 were postulated to be present in spring wheat cultivars. Seedling genes Lr3, Lr14a, and Lr23 likely were present in some cultivars but could not be clearly identified in this study. Most of the cultivars had some level of adult plant leaf rust resistance, most likely due to Lr34. Cultivars that had seedling resistance genes Lr1, Lr2a, Lr10, or Lr16 had poor to intermediate levels of leaf rust resistance in field plots. Cultivars with combinations of seedling resistance genes Lr16 and Lr24 with additional adult plant resistance were highly resistant to leaf rust.