This study examined the effect of hybrid and inoculant application on the chemical composition, fermentation indices and fatty acid profile of barley silage. Two Korean barley hybrids (Yuyeon, YU and Youngyang, YO) were harvested at dough stage, which were 24.9% and 27.1% of dry matter, respectively. The forages were chopped to 4–6cm lengths and treated with or without inoculant at the ratio of 1.2 × 10⁴ cfu/g of L. plantarum on fresh weight basis. Treatments had a 2 × 2 factorial arrangement with 4 replicates. Each replicate was ensiled in a 10-L mini silo for 100 d and analyzed for chemical composition and in vitro DM digestibility. Data were analyzed with a model including hybrid, inoculant and the interaction using the GLM procedure of SAS. The YU silage had greater (P < 0.01) ash, acid detergent fiber and in vitro dry matter digestibility and mold counts than YO silage, but lower (P < 0.01) lactate and acetate concentrations. For YU but not YO, inoculation decreased the NDF concentration (P = 0.002), lactic to acetic acid ratio (P = 0.004), lactic acid bacteria (P = 0.007) and yeast counts (P < 0.001) and increased the pH (P = 0.032) and ammonia-N concentration (P = 0.026) (hybrid × inoculant interaction). The YU silage had greater (P < 0.005) C14:0, C16:0, C18:3n-3 and saturated fatty acid concentrations than YO silage, but lower (P = 0.001) C18:2n-6 concentration. Inoculation decreased (P < 0.05) C18:3n-3 concentrations, but increased (P < 0.05) C18:2n-6 and saturated fatty acid concentrations. For YU but not YO, inoculation decreased total fatty acid (P = 0.034) and polyunsaturated fatty acid (P = 0.003) concentrations but increased C18:0 (P = 0.011), C18:1n-9 (P = 0.044), monounsaturated fatty acid (P = 0.033) concentrations (hybrid × inoculant interaction). The PCR analysis followed by gel electrophoresis showed higher concentration of L. plantarum DNA in the inoculated silage. In conclusion, YU silage was more digestible but it had greater mold counts than YO silage. Inoculation did not improve the fermentation but it reduced the acetate and 18:3n-3 fatty acid concentrations and increased the C18:2n-6 and saturated fatty acid concentration.