Leaf rust, caused by Puccinia triticina (Pt), is a serious constraint to wheat production. Developing resistant varieties is the best approach to managing this disease. Wheat leaf rust resistance (Lr) genes have been classified into either all-stage resistance (ASR) or adult-plant resistance (APR). The objectives of this study were to identify sources of leaf rust resistance in contemporary U.S. hard winter wheat (HWW) and to dissect the genetic basis underlying leaf rust resistance in HWW. A panel of 732 elite HWW genotypes was evaluated for response to U.S. Pt races at the seedling stage and at the adult plant stage in leaf rust nurseries in Oklahoma, Texas, and Kansas. Further, the panel was genotyped using Multiplex Restriction Amplicon Sequencing (MRA-Seq) and DNA markers linked to the known ASR genes Lr18, Lr19, Lr21, Lr24, Lr37, and Lr42 and APR genes Lr34, Lr46, Lr67, Lr68, Lr77, and Lr78. Single nucleotide polymorphism (SNP) markers derived from MRA-Seq, DNA markers linked to the known Lr genes, and the phenotypic data were used for genome-wide association study (GWAS) to identify markers associated with leaf rust response. Gene postulation based on leaf rust reactions, DNA markers, and GWAS suggested the presence of Lr1, Lr2a, Lr10, Lr14a, Lr16, Lr18, Lr19, Lr21, Lr24, Lr26, Lr34, Lr37, Lr39, Lr42, Lr46, Lr68, Lr77, and Lr78 in the HWW panel. The GWAS identified 59 SNPs significantly associated with leaf rust response, of which 20 were likely associated with novel resistance loci and can be used to enhance wheat leaf rust resistance.