Wheat leaf rust, caused by Puccinia triticina Eriks., is one of the most widespread and destructive diseases of wheat in Canada and worldwide. Commonly used control measures include host genetic resistance and fungicide application. Genetic resistance has proven to be very effective and can also be durable when the right combinations of resistance genes are incorporated into wheat cultivars. However, P. triticina is a highly variable pathogen with diverse populations over large geographic areas. This often leads to the evolution of virulence toward resistance genes that are widely used in wheat cultivars. These virulent races are then rapidly selected by wheat cultivars that depend on these now ineffective resistance genes for protection. It is critical to detect these virulent races as quickly as possible to alert producers when some cultivars which were formerly resistant are now susceptible so that they can switch to other, more resistant cultivars or chemical control options. Similarly, wheat breeders need to be alerted that certain resistance genes and cultivars have lost their effectiveness to enable them to select alternative resistance genes and gene combinations for which virulence has not been detected. The purpose of this chapter is to describe the detailed methods required to reliably determine the virulence phenotype, or race, for isolates of P. triticina.