The discovery of barley Mlo demonstrated that filamentous pathogens rely on plant genes to achieve entry and lifecycle completion in barley leaves. While having a dramatic effect on foliar pathogens, it is unclear whether overlapping or distinct mechanisms affect filamentous pathogen infection of roots. To remove the bias connected with using different pathogens to understand colonization mechanisms in different tissues, we have utilized the aggressive hemibiotrophic oomycete pathogen Phytophthora palmivora. P. palmivora colonizes root as well as leaf tissues of barley (Hordeum vulgare). The infection is characterized by a transient biotrophy phase with formation of haustoria. Barley accessions varied in degree of susceptibility, with some accessions fully resistant to leaf infection. Notably, there was no overall correlation between degree of susceptibility in roots compared with leaves, suggesting that variation in different genes influences host susceptibility above and below ground. In addition, a developmental gradient influenced infection, with more extensive colonization observed in mature leaf sectors. The mlo5 mutation attenuates P. palmivora infection but only in young leaf tissues. The barley-P. palmivora interaction represents a simple system to identify and compare genetic components governing quantitative colonization in diverse barley tissue types.