Posttranslational histone modifications play an important role in modulating gene expression and chromatin structure. Here we report the identification of histone H3K79 dimethylation in the simple eukaryote Dictyostelium discoideum. We have deleted the D. discoideum Dot1/KMT4 homologue and demonstrate that it is the sole enzyme responsible for histone H3K79me2. Cells lacking Dot1 are reduced in growth and delayed in development, but do not show apparent changes in cell cycle regulation. Furthermore, our results indicate that Dot1 contributes to UV damage resistance and DNA repair in D. discoideum. In summary, the data support the view that the machinery controlling the setting of histone marks is evolutionary highly conserved and provide evidence that D. discoideum is a suitable model system to analyze these modifications and their functions during development and differentiation.