Thatcher and 36 near-isogenic Thatcher lines with different Lr genes for resistance to Puccinia recondita f. sp. tritici were planted in field plots at one location each in the eastern and central parts of the forest-steppe region of Ukraine where they were exposed to natural epiphytotics of leaf rust. Disease severity on the lines indicated that Lr9, Lr19, Lr24, Lr25, and Lr28 had highly effective resistance in both locations. Lines with Lr27+Lr31, Lr35, and Lr36 were resistant in the eastern location and highly resistant in central location. Bulk collections of P. recondita from fields of nine Ukrainian winter wheat cultivars in the eastern location were inoculated onto seedlings of each of the 36 Thatcher near-isogenic lines. Lines with Lr9, Lr19, Lr24, Lr25, and Lr28 remained free or nearly free of uredinia. A total of 133 single-uredinial isolates obtained from 19 susceptible Thatcher near-isogenic lines in field plots in the central part of the forest-steppe region were identified to race using the eight standard leaf rust differential varieties of Johnson and Browder. The most common races were races 6 (27.8%), 77 (18.0%), 149 (13.5%), and X-4 (9.8%). The three most common races were not found on the Lr26 line and may lack virulence to it.