Differences in elm susceptibility against Ophiostoma novo-ulmi have been related with differences in timing and degree of the tree defence responses. In this study, we used Fourier transform-infrared spectroscopy, coupled with chemometric methods, to detect progressive changes in the metabolic profile of Ulmus minor and U. minor × U. pumila xylem tissues after inoculation with O. novo-ulmi. Differences between control and inoculated trees were detected at 30 and 60 days post-inoculation (dpi) in U. minor, and at 15, 30 and 60 dpi in U. minor × U. pumila. These differences were related with increased levels of lignin in the xylem tissues, suggesting an earlier defence response to the infection in the hybrids.