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Barley leaf rust (Puccinia hordei Otth.) is considered a harmful disease that occurs in barley-growing regions worldwide. In Russia, the disease is among the most prevalent in the Krasnodar region, which is the leader in the production of barley grain and has a favorable climate for disease development. In this paper, we studied the efficiency of 17 varieties and lines of barley from the International and Australian sets containing currently known Rph resistance genes or their combinations to P. hordei in the field, and 15 varieties and lines in the seedling phase in greenhouse conditions during 2019–2021. We concluded that the lines carrying the Rph7 and Rph13 genes remained immune throughout the three years of studies in the seedling and adult plant stages. The Rph1 and Rph23 genes showed moderate efficiency during the three years. The Rph2, Rph3, Rph4, Rph5, Rph6+2, Rph8, Rph12, Rph19, and Rph21+2 genes showed low efficiency over the three years. This was also confirmed by the results of their assessment in the seedling phase: the number of monopustular isolates virulent to lines with the majority of the studied genes for three years was above 90%. Fluctuations in the virulence of the P. hordei population were observed under sufficiently unfavorable weather for disease development in 2019, 2020, and 2021. This proves the ability of the fungus to adapt to changing conditions. Therefore, annual monitoring of the response of lines and varieties carrying resistance genes and studying the virulence of the pathogen are crucial for the selection of rust-resistant varieties, and, hence, the prevention of barley leaf rust epidemics in all grain-producing regions worldwide. [ABSTRACT FROM AUTHOR]