Most new coffee cultivars disseminated over the last 15 years are derived from the Timor Hybrid (Coffea arabica x C canephora). Introgression of genes from the C canephora genome has been estimated at between 9 and 29% of the genome. It has been shown that introgression can have a negative impact on the cup quality of cultivars derived from the Timor Hybrid. Consequently, coffee buyers or roasters may wish to assess whether the coffee they are purchasing comes from introgressed varieties. The possibility of distinguishing between non-introgressed Arabicas and genotypes carrying chromosome fragments introgressed from C canephora was investigated (i) using some classical chemical compounds (caffeine, chlorogenic acids, trigonelline, fat and sucrose) and (ii) using a new approach based on spectra acquired by near-infrared reflectance of green coffee. Near-infrared spectra were obtained for 129 samples from two collections (Nicaragua and Costa Rica) of introgressed and non-introgressed coffee trees. The spectral collections were treated by principal component and factorial discrimination. When the introgressed coffee trees were compared with the non-introgressed trees using the chemical compounds, small but significant differences were found in caffeine, trigonelline and chlorogenic acid contents. However, the small variations in those compounds are not enough to detect introgression. The spectral collections treated by principal component and factorial discrimination made it possible to class from 92.30 to 94.87% of the analysed samples correctly, while the percentages of correctly classified samples in the verification file varied from 88.23 to 94.11%. The NIRS method appears to be an efficient method for determining whether a green coffee comes from an introgressed variety.