Wheat leaf rust, caused by Puccinia triticina, is reported annually in southern Russia, the main wheat production region in Russia. The effect of biotic (types of cultivar resistance) and abiotic (northern, central and southern agro‐ecological zones) factors on virulence variability of the pathogen was analysed with 43 Lr differentials. Virulence frequencies to Lr genes 1, 2c, 3, 3bg, 11, 14a, 14b, 16, 17, 18, 23, 25, 26, 33, 34, 40 and Kanr exceeded 50% in all populations. A total of 81 virulence phenotypes (based on the standard set of 20 differentials) were identified among 108 isolates. The most common phenotypes were PHSTQ, THTTR, PHTTR and TKTTR. The minimum genetic distance (the highest similarity) was established between the P. triticina populations from susceptible cv. Krasnodarskaya 99 and cv. Brigada with race‐specific resistance. P. triticina populations from the susceptible cultivar in the three agro‐ecological zones were less variable and more similar than the cultivar‐specific and natural pathogen populations. Resistance types of host cultivars seem to have a greater effect on the pathogen population than abiotic factors. Nevertheless, abiotic factors need to be considered when making decisions about deployment of wheat varieties. Our results support the paradigm of a shift in the population of an obligate parasite towards increased virulence in response to the selection pressure of cultivars with race‐specific resistance.