Fusarium head blight (FHB), caused by Fusarium species, primarily F. graminearum is a destructive disease of barley (Hordeum vulgare L.) in many warm and humid growing regions of the world. Selection of barley varieties for FHB resistance and low DON accumulation is usually conducted in field nurseries or in controlled environments. However, the results obtained with the existing inoculation methods are highly variable and non-reproducible. This might be associated with the fact that the stage at which FHB infection takes place, primarily through anthers, happens while the anthers are still enclosed in the palea. Exposing the anthers by cutting the tip of the developing kernel at heading/early anthesis significantly increased disease infection rate most probably due to an increased chance of direct contact between spores and anthers during inoculation. Inoculation of barley lines previously reported as resistant following the standard spray inoculation procedure (kernels intact) resulted in a resistant reaction with low FHB severity. However, when the tip of the kernel/palea of these lines were excised and the macroconidia spray-inoculated on exposed anthers, FHB severity dramatically increased, and the lines became completely susceptible. The DON content on excised kernels was also significantly higher than on intact kernels. This suggests that the resistance observed on plants inoculated on intact palea may be due to physical barrier (of spores from coming in to contact with the anther) rather than genetic. The clip inoculation method may be a reliable method to confirm true genetic resistance of barley to FHB. Further investigation including gene expression analysis is required to fully understand this phenomenon.