Abstract Tsetse flies ( Diptera: Glossinidae ) are the cyclical vectors of trypanosomes, the causative agents of African animal trypanosomosis or nagana in animals and human African trypanosomosis or sleeping sickness in humans. Tsetse flies are K-strategist species with the deposition of a single larva at 10 days intervals in specific sites. As larviposition site selection will strongly impact reproductive success, it is obvious that the selection of larviposition sites is not random and is under strong selective processes, probably mediated by specific cues as suggested by the existence of an aggregation factor in the Morsitans and Fusca groups. This study aimed to highlight the existence of an aggregation effect in the Palpalis group and to test for its chemical nature. We studied the larviposition site selection of Glossina palpalis gambiensis according to the presence of conspecific and heterospecific larvae buried in substrates in different settings. Three sets of experiments were performed with either individual or grouped (n = 50) gravid females, and with physical access to substrate or not. In both individual and grouped larviposition experiments, females selected significantly more often trays conditioned by larvae (P<0.005), either conspecific or heterospecific even in the absence of physical contact with the substrate. These results highlight the first evidence for larviposition site selection mediated by volatile semiochemicals of larval origin in Glossina palpalis gambiensis . However, these compounds seem not to be species-specific and therefore offer new avenues for the behavioural manipulation of these vectors and for the development of new vector control tools targeting gravid females. Author summary Larviposition site selection in tsetse flies is govern by several biotic and abiotic factors that lead to an aggregation effect of larvae. Among those, larvae are suspected to produce chemicals that drive females to breeding site but little information is available. This study aimed to highlight the existence of an aggregation effect of larval origin in the Palpalis group and to test for its chemical nature. Through behavioural larviposition choice experiments, we showed that females of Glossina palpalis gambiensis deposit their larvae significantly more often in trays conditioned either by conspecific or heterospecific larvae, even in the absence of physical contact with the substrate. These results highlight the first evidence for aggregation effect in Glossina palpalis gambiensis mediated by volatile semiochemicals of larval origin. Isolation and identification of these chemicals should offer new avenues for the behavioural manipulation of these vectors and for the development of new vector control tools targeting gravid females.