Knockout mice lacking serotonin1B(5‐hydroxytryptamine1B; 5‐HT1B) receptors (1BKO) exhibit increased sensitivity to the stimulant and reinforcing effects of indirect dopamine (DA) agonists and are more reactive to mild stressors such as handling and the injection procedures that commonly occur when using the intraperitoneal (i.p.) route of drug administration. Since the intravenous (i.v.) route of administration allows minimal handling and injection‐induced stress, the present study was designed to evaluate the effect of the administration route on amphetamine‐induced locomotor activation and behavioural sensitization in 1BKO mice. For this purpose, 1BKO and wild‐type (WT) control mice were administered i.p. or i.v. amphetamine in a within‐session design, which allows evaluation of a complete dose–response curve within a single session. The locomotor stimulant effects of i.p. (0.5–4.0 mg/kg) and i.v. (0.6–1.2 mg/kg) amphetamine were investigated both acutely and following repeated treatments (four treatments at 48 h intervals). The results showed that acute i.p. amphetamine injection induced a significant higher horizontal activity peak effect in 1BKO mice, while this difference was less profound after acute i.v. injection. However, repeated i.p. or i.v. administration of amphetamine induced significantly higher locomotion in 1BKO mice. We conclude that the stimulant effects of amphetamine can be influenced by the route of administration in a genotype‐dependent manner, and that route of drug administration (and associated variables) should be considered an important factor in studies of psychostimulant action in knockout mice.