Spot blotch caused by ascomycete fungus Cochliobolus sativus (Ito & Kurib.) Drechs. ex Dastur. [anamorph: Bipolaris sorokiniana (Sacc.) Shoem.] is one of the most common and economically important diseases on barley. To better understand the molecular interaction between the different pathotypes of this pathogen and barley differential lines, fungal genes involved in virulence and barley genes for spot blotch resistance were characterized in this study. Previous studies have revealed that the virulence factor in the pathotype 2 isolate ND90Pr making cv. Bowman susceptible is a secondary metabolite peptide synthesized by two non-ribosomal peptide synthesdases (NRPSs). However, the global regulation of biosynthesis of this secondary metabolite is not well understood in this fungus. Recently, the velvet-complex proteins containing LaeA and velvet proteins (VeA, VelB, VelC and VosA) have been shown to be involved in global regulation of secondary metabolism and fungal development in many fungal pathogens. To characterize the functions of the orthologous of velvet-complex proteins in C. sativus, single and double gene knockout mutants were generated. The results indicated that the velvet-complex factors affect virulence of the fungus by regulating expression of the NRPSs involved in virulence. In addition, velvet-complex proteins were found to coordinately and distinctly regulate fungal development, such as conidiogenesis and conidia germination. To identify and characterize genes for resistance to the new pathotype, 2,062 barley accessions from a USDA barley core collection were screened for spot blotch resistance to this pathotype and 24.5% of them showed resistance or moderate resistance at the seedling stage. Genome-wide association analysis identified four QTLs associated with the seedling resistance, which were located on chromosome 1H, 2H, 3H, and 6H, respectively. A genetic analysis of the cross between a highly resistant line (PI 235186) and a highly susceptible accession (PI 356741) suggested a single dominant gene confers the resistance in PI 235186. The resistance gene was further mapped to the short arm of chromosome 6H based on bulk segregant analysis using 194 SSR markers and genotyping-by-sequencing using 20 SNP markers in a F2 population. Additional markers were developed to fine map the resistance gene to a ~6.5 cM genomic region.