Pumpkin (Cucurbita maxima) seeds have gained increasing attention due to their high nutraceutical value; however, data regarding the phospholipid (PL) profile of this oily seed are scarce. Through the combination of RPLC-MS/MS, HILIC-MS, and Paternò-Büchi photochemical reaction, we comprehensively characterized PLs in three accessions of pumpkin seeds. We identified a total of 183 PL molecular species across eight classes. Phosphatidylcholine (PC) represents the most abundant class, followed by phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA). The total PL content is comparable to that of almonds and cashew nuts. In terms of PL structures, pumpkin seeds feature a higher abundance of C18:2-containing PL isomers, a preferential esterification of C18:1 and C18:2 at the sn-2 position as well as a predominant Δ9/Δ9,12 double bond locations for C18:1 and C18:2 respectively. This is the first systematic study on PLs of pumpkin seeds to our knowledge. Our findings add further evidence on the potential value of pumpkin seeds as a functional food or as a source material for making plant-based drink.