Wheat yellow rust (Puccinia striiformis f. sp. tritici West. PST) is one of the main fungal diseases that causes major yield losses in the central and southern wheat production area of Chile. Chilean plant breeding programs have been using specific resistance genes to control this disease and resistance was frequently lost due to the appearance of new pathotypes of this pathogen. This resulted in a frequent change of wheat cultivars in the country. The objective of this work was to determine the genetic polymorphism among PST samples collected in wheat fields throughout the country, using randomly amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP). Twenty-nine PST populations from 5 locations were analysed with 64 RAPD primers and 6 combinations of AFLP primers. The coefficients of similarity (53% for RAPD and 14% for AFLP) indicated that the populations of yellow rust in Chile are highly diverse, in contrast with similar studies performed in other countries. This observation is supported by the virulence spectrum of PST population. This stressed the need to pyramid 2 or more major genes in commercial cultivars. Alternatively, the deployment of adult plant resistance gene combinations should be conducted. AFLP technique was more robust for analysis of the genetic diversity of PST compared with RAPD, because of its reproducibility and high level of polymorphism. The analysis of genetic similarity data among yellow rust populations grouped most of the PST population according to their geographic origin.