Nestmate recognition is a necessary capacity for the occurrence of discrimination between nestmate and non-nestmate individuals. In one-piece nesting termites, which nest and forage in a single piece of wood, nestmate recognition is poorly studied mainly because the probability of encountering exogenous individuals is low in comparison with separate-piece nesting termites. Previous work described that production of soldiers of Neotermes chilensis, a one-piece nesting termite, increased when the risk of invasion of their colony increased, for example when neighboring colonies were present in the same nesting substrate and members of different colonies met when digging galleries. If soldiers are to fulfill their defensive role under these circumstances, they should show nestmate recognition ability; moreover, based on work on other social insects, such nestmate recognition should be based on cuticular compounds (CC). Bioassays were performed in which a soldier of N. chilensis was confronted with a nestmate or non-nestmate primary reproductive, pseudergate or another soldier, and in which a soldier was confronted with untreated and with CC-deprived dead primary reproductives. The results showed that soldiers were indeed more aggressive toward non-nestmates than nestmates for all castes, and that this discrimination was mediated mainly by qualitative (simple matching coefficient) and quantitative (Renkonen index) differences in CC.