Vendor differences are thought to affect Pavlovian conditioning in rats. After observing possible differences in sign-tracking and goal-tracking behaviour with rats from different breeding colonies, we performed an empirical replication of the effect. 40 male Long-Evans rats from Charles River colonies ‘K72’ and ‘R06’ received 11 Pavlovian conditioned approach training sessions (or “autoshaping”), with a lever as the conditioned stimulus (CS) and 10% sucrose as the unconditioned stimulus (US). Each 58-min session consisted of 12 CS-US trials. Paired rats (n = 15/colony) received the US following lever retraction. Unpaired control rats (n = 5/colony) received sucrose during the inter-trial interval. Next, we evaluated the conditioned reinforcing properties of the CS, by determining whether rats would learn to nose-poke into a new, active (vs. inactive) port to receive CS presentations alone (no sucrose). Preregistered confirmatory analyses showed that during autoshaping sessions, Paired rats made significantly more CS-triggered entries into the sucrose port (i.e., goal-tracking) and lever activations (sign-tracking) than Unpaired rats did, demonstrating acquisition of the CS-US association. Confirmatory analyses showed no effects of breeding colony on autoshaping. During conditioned reinforcement testing, analysis of data from Paired rats alone showed significantly more active vs. inactive nosepokes, suggesting that in these rats, the lever CS acquired incentive motivational properties. Analysing Paired rats alone also showed that K72 rats had higher Pavlovian Conditioned Approach scores than R06 rats did. Thus, breeding colony can affect outcome in Pavlovian conditioned approach studies, and animal breeding source should be considered as a covariate in such work.Vendor differences are thought to affect Pavlovian conditioning in rats. After observing possible differences in sign-tracking and goal-tracking behaviour with rats from different breeding colonies, we performed an empirical replication of the effect. 40 male Long-Evans rats from Charles River colonies ‘K72’ and ‘R06’ received 11 Pavlovian conditioned approach training sessions (or “autoshaping”), with a lever as the conditioned stimulus (CS) and 10% sucrose as the unconditioned stimulus (US). Each 58-min session consisted of 12 CS-US trials. Paired rats (n = 15/colony) received the US following lever retraction. Unpaired control rats (n = 5/colony) received sucrose during the inter-trial interval. Next, we evaluated the conditioned reinforcing properties of the CS, by determining whether rats would learn to nose-poke into a new, active (vs. inactive) port to receive CS presentations alone (no sucrose). Preregistered confirmatory analyses showed that during autoshaping sessions, Paired rats made significantly more CS-triggered entries into the sucrose port (i.e., goal-tracking) and lever activations (sign-tracking) than Unpaired rats did, demonstrating acquisition of the CS-US association. Confirmatory analyses showed no effects of breeding colony on autoshaping. During conditioned reinforcement testing, analysis of data from Paired rats alone showed significantly more active vs. inactive nosepokes, suggesting that in these rats, the lever CS acquired incentive motivational properties. Analysing Paired rats alone also showed that K72 rats had higher Pavlovian Conditioned Approach scores than R06 rats did. Thus, breeding colony can affect outcome in Pavlovian conditioned approach studies, and animal breeding source should be considered as a covariate in such work.