In Hordeum chilense Roem. & Schult., a high variation in the level of avoidance to infection of barley leaf rust (Puccinia hordei Otth) occurs. Probably resulting from the properties of the stomata, the rust germ tube overgrows stomata, and the infection process fails in an early stage. In the present study we tested the hypothesis that the avoidance character occurs in certain morphologically and molecularly distinct ecotypes of H. chilense. Eighty-eight H. chilense accessions were inoculated with P. hordei to assess the level of avoidance. The accessions were described for 30 morphological characters and three AFLP primer combinations. Cluster analysis using both morphological and AFLP fingerprint data suggested three distinct clusters of accessions. One of the clusters had a particularly high level of avoidance. This putative subspecific taxon was characterized by shorter and wider spikes, more erect culms, a greater number of stomata per square centimetre on the abaxial leaf side, and a shorter uppermost internode until flag leaf. All accessions clustered in this subspecific taxon were collected from humid habitats. We conclude that H. chilense consists of at least three rather well defined, morphologically and genetically distinct subspecific taxa, one of which has a very high level of avoidance to barley leaf rust.