Cochliobolus sativus is the causal agent of several important cereal diseases, including spot blotch, common root rot, and black point. To establish a high throughput gene silencing system for functional genomics studies of the fungus, an RNAi vector based on the Gateway Cloning system was constructed. The RNAi system was further demonstrated by knocking down CsPKS1, which indicated that the established RNAi system is a useful tool for characterization of gene functions in C. sativus. To develop DNA markers for genetic study, a total of 162 putative SSR loci were identified and flanking primers were developed for PCR amplification. Fifty-one of the loci were polymorphic between two parental isolates (ND93-1 and ND90Pr) and 43 loci showed the expected 1:1 segregation ratio in the mapping population. Twenty-two of the 43 SSR loci were positioned on the existing genetic map of C. sativus on 15 of the 27 linkage groups and 8 were mapped on 5 new linkage groups. A subset of 14 SSR markers selected from each linkage group was used to study the genetic diversity of C. sativus isolates. Phylogenetic analysis with these markers revealed that isolates from barley were separated from isolates from wheat. To characterize genes involved in fungal development, pathogenicity and virulence, the orthologues of MAP kinase genes ( CHK1, MPS1 and HOG1), MAPKK kinase gene (STE11) and a sfp-type-4'-phosphopantetheinyl transferase gene (PPT1) in C. sativus were identified by BLAST search against a draft genome sequence of the C. sativus strain ND90Pr. Deletion mutants were generated for each of the genes and characterized for phenotypic changes. The results indicated that all these MAP kinase and MAPKK kinase genes contribute to the regulation of fungal development under normal and stress conditions and are required for full virulence on the barley plants. The PPT1 gene is required for lysine synthesis, tolerance to oxidative stress, and virulence in C. sativus. It is hypothesized that an unknown virulence factor, presumably synthesized by PKSs or NRPSs requiring activation by PPT1, is directly responsible for high virulence on barley cv. Bowman in C. sativus.