Technical Abstract: Leaf rust resistance gene Lr21 is present in hard red spring wheat (Triticum aestivum) cultivars grown in Minnesota, North Dakota, South Dakota, Manitoba and Saskatchewan. Lr21 was originally derived from the wild wheat relative T. tauschii. Isolates of Puccinia triticina, the causal organism of wheat leaf rust, with virulence to Lr21 have not been previously detected in annual virulence surveys in the U.S. In 2010 the hard red spring wheat cultivars Faller, RB07, and Glenn all with Lr21 had higher levels of leaf rust severity in research plots than in previous years in North Dakota and Minnesota. Leaf rust collections from wheat cultivars and germplasm lines with Lr21 at three locations were increased on plants of Thatcher wheat and the Thatcher line with Lr21. Single uredinia from the collections were isolated and increased on seedlings of Thatcher. The single uredinial isolates were inoculated to 7-8 day old seedling plants of the set of 19 differential lines that are currently used in the leaf rust virulence surveys and four additional lines. The isolates were also inoculated to seedling plants of hard red spring wheat cultivars with Lr21: Glenn, Steele, Faller, RB07, Amidon, AC Cora, and McKenzie. Two virulence phenotypes, TFBJQ and TFBGQ with virulence to Lr21 were found at the three locations. TFBJQ is virulent (infection types 3-4) to genes Lr1, 2a, 2c, 3, 24, 26, 10, 14a, 21, 28, 14b, 20 and avirulent (infection types 0- 2+) to genes Lr9, 16, 3ka, 11, 17, 30, B, 18, 3bg, and Lr39/41. TFBGQ was avirulent to Lr14a and Lr20, and identical to TFBJQ for virulence and avirulence to the other resistance genes. Isolates of both phenotypes were virulent on Faller, Glenn, RB07, Steele, AC Cora and Amidon. McKenzie had infection type of 2+ due to the additional presence of Lr16. Both TFBJQ and TFBGQ have intermediate infection type of 2+ to Lr16; infection type 2+3 to Lr23, and are completely virulent to genes Lr1, Lr2a, and Lr10 that are present in hard red spring wheat cultivars. Both phenotypes are also virulent to genes Lr24 and Lr26 that are present in soft red winter wheat and hard red winter wheat cultivars. The Lr21 virulent phenotypes likely arose by mutation from the group of P. triticina genotypes in the simple sequence repeat group NA-5. The detection of P. triticina isolates with virulence to Lr21 represents a new threat to wheat production since in 2010 over 50% of the hard red spring wheat acreage in Minnesota and North Dakota relied on Lr21 for effective resistance to leaf rust.