The intact yeast phase spores of Ophiostoma ulmi and O. novo‐ulmi isolates were analysed by matrix‐assisted‐laser‐desorption/ionization time‐of‐flight/time‐of‐flight mass spectrometry. The results clearly showed that isolates of O. ulmi and O. novo‐ulmi have different mass peak profiles. The specific mass in m/z may vary slightly. One of the peaks from m/z 7623 to 7630 dominated (in high intensity) mass spectra for all isolates of O. novo‐ulmi tested in this study. This specific peak is believed to be CU (cerato‐ulmi) protein. However, no specific m/z peak can be found to distinguish between the isolates of ssp. novo‐ulmi and ssp. americana. On the contrary, all isolates of O. ulmi tested possessed one of a dominate peak at m/z 7337 to 7342, but none around m/z 7626 as shown in O. novo‐ulmi isolates.