Medicago truncatula lines resistant (A17) or susceptible (F83005.5) to the alfalfa pathogen Colletotrichum trifolii were used to compare defense reactions induced upon inoculation with C. trifolii or with the nonadapted pathogens C. lindemuthianum and C. higginsianum. Nonadapted Colletotrichum spp. induced a hypersensitive response (HR)-like reaction similar to the one induced during the host-incompatible interaction. Molecular analyses indicated an induction of PR10 and chalcone synthase genes in host and nonhost interactions but delayed responses were observed in the F83005.5 line. The clste12 penetration-deficient C. lindemuthianum mutant induced an HR and defense gene expression, showing that perception of nonadapted strains occurs before penetration of epidermal cells. Cytological and transcriptomic analyses performed upon inoculation of near-isogenic M. truncatula lines, differing only at the C. trifolii resistance locus, Ct1, with the nonadapted Colletotrichum strain, showed that nonhost responses are similar in the two lines. These included a localized oxidative burst, accumulation of fluorescent compounds, and transient expression of a small number of genes. Host interactions were characterized by a group of defense and signaling-related genes induced at 3 days postinoculation, associated with an accumulation of salicylic acid. Together, these results show that M. truncatula displays a rapid and transient response to nonadapted Colletotrichum strains and that this response is not linked to the C. trifolii resistance locus.