Three different routes of Foot-and-mouth disease virus (FMDV) infection of piglets, namely intranasal (i.n.) through drops, intradermal (i.d.) into the foot, and intramuscular (i.m.) were compared regarding the onset and severity of the disease. The results showed that the i.d. injection of the virus resulted in the fastest onset of the disease. The i.m. injection led to a delayed onset, but the final effect was identical with i.d. injection. Moreover, the i.m. injection was simpler to perform and easier to evaluate. Therefore, the i.m. injection of piglets is recommended as the optimal infection route for evaluation of the FMDV vaccine potency.