Leaf rust is the most widespread and frequently occurring fungal disease of wheat (Triticum aestivum L.) in Ukraine and worldwide. The information about the effectiveness of Lr-genes and also the consequent monitoring of virulence dynamics is necessary for the successful wheat breeding for leaf rust resistance. In 2013-2014 pathotype composition and virulence analysis was studied both on the standard differential set and on the North American System of Nomenclature. According to the standard differential set, 12 phenotypes were identified, of which the most common were 77 (75%) and 144 (6%). A total of 40 phenotypes were identified on the North American Nomenclature. Phenotypes TGTT (24%) and TJTT (8%) were the most frequent, TRTT (1.5%) and TSTT (1.5%) were within the broadest spectrum of virulence among the isolates found in the south of Ukraine. For virulence analysis we used wheat lines of ’Thatcher’ that are near-isogenic for 24 leaf rust resistance genes and additionally four cultivars/lines. No virulence to Lr19 was found, whereas increasing virulence to Lr9 was detected (13%). Low frequency of virulence was observed to Lr29 (11%) and Lr47 (21%), high level of virulence was detected to other genes. The effectiveness of 53 known Lr-genes was studied at the seedling and the adult plant stages. Most of them were not effective against leaf rust. Genes Lr9, Lr19, Lr29, and Lr47 were highly effective both at the seedling stage and at adult plant stage. Genes Lr24, Lr42, Lr50, Lr51, and Lr56 were effective only at the adult plant stage.