This paper reports on the identification of new diketopiperazines (DKPs) in roasted coffee. The roasted coffee was extracted with hot water and CHCl3 to achieve both a cleanup and a concentration of the DKPs. To get rid of the huge caffeine peak in both HPLC and GC analysis, this extract was cleaned up again by ion-exchange chromatography on Dowex 50×8 followed by gel chromatography on Sephadex G10. The identification was accomplished by HPLC-ESI-MS, HPLC-ESI-MS/MS, GC-EI-MS and by comparison with synthesised reference compounds. In roasted coffee the following DKPs were identified: cyclo(pro-gly), cyclo(pro-ala), cyclo(phe-val), cyclo(phe-leu), and cyclo(phe-ile). Except for cyclo(pro-gly), where only one isomer can be formed, each DKP was present in both possible isomeric forms.