Fusarium head blight (FHB), mainly caused by the fungus Fusarium graminearum, is one of the most destructive diseases of barley, wheat and other small cereals in the world. Intensive efforts on screening FHB resistance in barley have not been fruitful yet.  The RNA interference (RNAi) approach has been successfully used to control various plant pathogens. However, the application of RNAi to enhance barley FHB resistance is limited. We constructed vectors containing the fragmented Tri6 gene, a transcriptional regulator within tricothecene gene cluster of Fusarium graminearum and transferred the vectors into a malting barley variety Germcraft using the meristem transformation method. We generated 15 T0 transgenic plants including five generated on hygromycin selection and 10 on G418 selection. The T1 seeds were planted and grown for PCR analysis. Expected bands were amplified from 10 T1 lines. Droplet digital PCR (ddPCR) with the T1 transgenic plants identified lines with various copies of the transgene. Nearly 200 T2-generation plants were analyzed by PCR and ddPCR, and five homozygous T2 lines with single copy transgene were identified. Some of the T2 plants were phenotyped through DIP inoculation with a most virulent Fusarium graminearum strain PH1. Significant variations in both disease severity and deoxynivalenol (DON) accumulations were observed among the T2 transgenic plants. We plan on conducting large-scale FHB phenotyping and expression of small RNAs of Tri6 gene with T3 stable transgenic lines.