In this study, we were interested in comparing the amino acid profile in a specific variety of onion, Rossa da inverno sel. Rojo Duro, produced in two different Italian sites: the Cannara (Umbria region) and Imola (Emilia Romagna region) sites. Onions were cultivated in a comparable manner, mostly in terms of the mineral fertilization, seeding, and harvesting stages, as well as good weed control. Furthermore, in both regions, the plants were irrigated by the water sprinkler method and subjected to similar temperature and weather conditions. A further group of Cannara onions that were grown by micro-irrigation was also evaluated. After the extraction of the free amino acid mixture, an ion-pairing reversed-phase high performance liquid chromatography-evaporative light scattering detector (IP-RP HPLC-ELSD) method allowed for the separation and detection of almost all the standard proteinogenic amino acids. However, only the peaks corresponding to leucine (Leu), phenylalanine (Phe), and tryptophan (Trp), were present in all the investigated samples and they were unaffected from the matrix interfering peaks. The use of the beeswarm/box plots revealed that the content of Leu and Phe were markedly influenced by the geographical origin of the onions (with *** p << 0.001 for Phe), but not by the irrigation procedure. The applied HPLC method was validated in terms of the specificity, the linearity (a logarithm transformation was applied for the method linearization), the limit of detection (LOD) and limit of quantification (LOQ), the accuracy (≥90% for inter-day Recovery percentage), and the precision (≤10.51 for the inter-day RSD percentage), before the quantitative assay of Leu, Phe, and Trp in the onion samples. These preliminary findings are a good starting point for considering the quantity of the specific amino acids in the Rossa da inverno sel. Rojo Duro variety as a fingerprint of its geographical origin.