Leaf rust caused by Puccinia triticina (Pt) is a major disease of bread wheat, particularly in the winter rainfall wheat growing regions of South Africa (SA). Studying the virulence profiles of the Pt population is crucial for effective management of leaf rust using genetic resistance. Organized national rust surveys have been conducted in SA for more than 40 years. To determine the phenotypic diversity of Pt, infected wheat leaves were collected between 2017 and 2020 from commercial wheat fields and rust trap nurseries across the major wheat growing regions of SA. Pt races were identified based on their avirulence/virulence profiles on seedlings of 16 standard differential lines. Nine races were identified from 366 isolates that were successfully pathotyped. Races CFPS, CDPS, MCDS and CBPS were most commonly found with average frequencies varying from 18% (MCDS and CBPS) to 27% (CFPS). The frequency of MFPS was about 10% whereas the remaining four races were observed at less than 3% frequency. Two new races (MFPS and MCPS) were identified during this study. MFPS, which is virulent on the key resistance genes Lr1, Lr24, Lr26, was first detected in 2020. The virulence profile of MFPS is related to the existing races CFPS, CDPS and MCDS. Race MFPS differs in virulence from CFPS on Lr1, from CDPS on Lr1 and Lr26 and from MCDS on Lr3ka, Lr24 and Lr30. From the phenotypic data, it appears that race MFPS may have developed locally through a single step mutation from race CFPS by gaining virulence for Lr1. Race MCPS was first detected in 2017. Except for its virulence on Lr3ka and Lr30, MCPS is similar in its virulence profile to the existing race MCDS. Seedling infection type data for 103 South African wheat varieties revealed that 35 of the entries are susceptible to races CBPS and MCPS, followed by race MCDS (38 susceptible), MFPS (51) and CFPS (77). With virulence to Lr24 and Lr26, races CFPS and MFPS are considered to remain prevalent and threatening compared to the other races. Microsatellite analysis revealed a close genetic relationship between races MCPS, MFPS and race MCDS. With the detection of MCPS and MFPS, the number of new Pt races reported over the past 10 years increased to nine, which means that on the average, about one new race has been identified every year. The results indicate continued variability of the Pt population in SA and underscore the need for regular surveillance to timely detect and use new races to screen and identify effective sources of resistance.