The temporal order in which experiences occur can have a profound influence on their salience. Humans and other vertebrates usually memorise the first and last items of a list most readily. Studies on serial position learning in insects, mainly in bees, showed preference for last encountered items. In bees, pheromone presence can also influence motivation, and thus learning. However, neither serial position learning nor the effect of recruitment pheromones on learning have been well investigated in ants. We trained Lasius niger ants to make multiple visits to sucrose on a runway which alternated between lemon or rosemary odour, and the presence or absence of trail pheromone, and then tested for preference between the odours on a Y-maze, in order to investigate the effect of pheromone presence on learning. Pheromone presence did not affect ant choice. However, unexpectedly, the ants strongly preferred the first odour encountered. This was explored by the addition of a familiarisation visit without pheromone or odour. The familiarisation visit disabled or reversed this preference for the first odour encountered, with ants now mostly taking their 'default' preference by choosing the left side of the maze. Our study found no effect of trail pheromone on learning, but a strong yet fragile preference for the first odour experienced. These different preferences could lead to spatial segregation of foraging activity depending on prior experience and might facilitate efficient resource exploitation by colonies.