In the frame of a broader project, we were interested at comparing the amino acid profile in a specific variety of onion, Rossa da inverno sel. Rojo Duro, produced in two different Italian sites: Cannara (Umbria region) and Imola (Emilia Romagna region). In both places, onions were cultivated and harvested in the same way, and irrigated by water sprinkler method. A further group of Cannara onions, growth by microirrigation, was also evaluated. After the extraction of free amino acid mixture from onion samples, an ion-pairing RP-HPLC method allowed the separation and the evaporative light scattering detection of almost all underivatized proteinogenic amino acids. However, only the peaks corresponding to Leu, Phe, Trp, were present in all the investigated samples and unaffected from matrix interfering peaks. The application of the beeswarm/box plots with the ANOVA/TukeyHSD statistical approach revealed a content of Leu and Phe markedly influenced by the geographical origin of the onions, while not by the irrigation procedure. The developed HPLC method was validated in terms of specificity, linearity, LOD and LOQ, accuracy and precision, before the quantitative assay of Leu, Phe and Trp in the onion samples. Although further studies are necessary, these preliminary findings can represent a good starting point for considering the quantity of specific amino acids in the Rossa da inverno sel. Rojo Duro variety as a fingerprint of its geographical origin. In principle, the developed approach might be applied to other onion varieties, thus contributing to their characterization and traceability, also contributing to limit commercial frauds.