In 2016 – 2018 virulence of the Czech wheat leaf rust population was studied on Thatcher near-isogenic lines carrying different Lr genes and 130 isolates were analysed. Virulence to Lr9 was found only sporadically. Virulence frequency to Lr2a, Lr2b, Lr2c and Lr28 was lower than in previous years. All tested isolates were avirulent to Lr19. Lr24 conditioned resistance to majority of isolates. Nineteen recently registered Czech cultivars were tested with six isolates of the pathogen and Lr genes were postulated. To support the gene postulations, presence of genes Lr1, Lr10, Lr19, Lr24, Lr26, Lr28, Lr34 and Lr37 was tested by molecular markers. Lr37 prevailed, followed by Lr genes 10, 24, 28, 1 and 26; genes Lr19 and Lr34 were not determined.