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Alfalfa leaf rust caused by the fungal pathogen Uromyces striatus compromises forage yield and quality. We investigated the genetic basis of leaf rust resistance in alfalfa using an F1 mapping population segregating for resistance to the disease in addition to segregation for fall dormancy (FD) and winter hardiness (WH). The objective of this work was to map quantitative trait loci (QTL) associated with alfalfa leaf rust resistance using genotyping-by-sequencing (GBS) SNP markers in the biparental F1 mapping population. The female parent 3010 displayed susceptibility to rust while the male parent CW 1010 was moderately resistant. The mapping population consisted of 184 F1 progenies that were planted with the parents in a randomized complete block (RCBD) design with three replications at Watkinsville and Blairsville, GA. Four clones generated from stem cuttings of each genotype were planted in each replication. Phenotyping rust severity (RS) was based on the NAAIC visual rating scores of 1–5. RS and FD exhibited a negative phenotypic correlation (r = −0.26). RS and WH also exhibited a negative correlation (r = −0.27) suggesting that the dormant and winter hardy genotypes were more susceptible to leaf rust than the non-dormant and cold sensitive. Four QTL for alfalfa leaf rust resistance and one QTL for rust susceptibility were identified in the CW 1010 genetic map while three QTL for rust susceptibility were identified in the 3010 parent. The most important QTL Us-RustR1 explained 13% (R2 = 0.13) of the phenotypic variance. This study suggests that leaf rust resistance in alfalfa is most likely incomplete and has polygenic inheritance. Comparative genome analysis of QTL regions using sequences of significant SNPs revealed homology to M. truncatula sequences that were previously reported for their role in plant defense mechanisms. Validation of the QTLs in different genetic backgrounds as well as fine mapping of the QTL regions would be the next step of this research. The markers presented in this study would be valuable resources for alfalfa genetic improvement via marker-assisted selection (MAS). • Eight QTLs associated with alfalfa rust resistance were detected using GBS of a pseudo-testcross mapping population. • In this population, the inheritance of rust resistance caused by Uromyces striatus was polygenic and incomplete. • The sequences of QTL SNPs exhibited homology with genes associated with fungal diseases defense in M. truncatula. • Alfalfa rust severity correlated with FD and WH, but the relationship might be specific to the population. [ABSTRACT FROM AUTHOR]