Hemp seed oil (HSO) has received considerable attention for its health properties, especially due to unsaturated fatty acid (UFA) content. In this work, the triacylglycerol (TAG) fraction of Italian and Extra-European HSO was characterized by applying an enzymatic approach, based on the use of pancreatic lipase and sn-1,2-diacylglycerol kinase. This procedure allows determination of the intrapositional FA% composition of TAG. The results of the stereospecific analysis are useful for deepening knowledge on HSO nutritional aspects. The high percentage of UFA (88.3–89.9%), in particular essential FA (74.4–85.9%), of HSO samples in sn-2 position is important for long-term health effects, but also to enhance the use of this oil as a functional ingredient in food, cosmetic and nutraceutical fields. Furthermore, the results of total and intrapositional FA % compositions, subjected to principal component analysis, were able to differentiate HSO Italian samples from Extra-European ones. Based on the obtained results, it can be stated that the stereospecific analysis represents a potent analytical tool providing the fingerprint of TAG fraction, useful to highlight possible chemical descriptors for HSO authenticity and traceability purposes.