Abstract Background Herbivore-induced defence responses are often specific - different herbivores induce different defence responses in plants - and their specificity is largely mediated by chemical cues (herbivore-associated elicitors: HAEs) in insect oral or oviposition secretions. However, the specificity and the mechanisms of HAE-induced defence have not been investigated in the context of the evolutionary relationships among plant species. Here we compare the responses of six closely related Nicotiana species to a synthetic elicitor, N -linolenoyl-glutamic acid (C18:3-Glu) and HAE of two insect herbivores (the Solanaceae specialist Manduca sexta and generalist Spodoptera littoralis ). Results HAE-induced defences are highly specific among closely related Nicotiana species at three perspectives. 1) A single Nicotiana species can elicit distinct responses to different HAEs. N. pauciflora elicited increased levels of JA and trypsin proteinase inhibitors (TPI) in response to C18:3-Glu and the oral secretions of M. sexta (OS Ms ) but not to oral secretions of S. littoralis (OS Sl ). In contrast, N. miersii only responded to OS Sl but not to the other two HAEs. The specific responses to different HAEs in Nicotiana species are likely due to the perception by the plant of each specific component of the HAE. 2) One HAE can induce different defence responses among closely related Nicotiana species. OS Ms and C18:3-Glu induced JA and TPI accumulations in N. linearis , N. attenuata , N. acuminata and N. pauciflora , but not in N. miersii and N. obtusifolia . 3) The effect of HAE-induced defences differ for the Solanaceae specialist M. sexta and the generalist S. littoralis . Among the four tested Nicotiana species, while the growth rate of M. sexta was only reduced by the induced defences elicited by C18:3-Glu; the growth rate of S. littoralis can be reduced by the induced defences elicited by all three HAEs. This is likely due to differences in the susceptibility of the specialist M. sexta and generalist S. littoralis to induced defences of their host. Conclusions Closely related Nicotiana species elicit highly specific defence responses to herbivore associated elicitors and provide an ideal framework for investigating the molecular mechanisms and evolutionary divergence of induced resistance in plants.