Nowadays there are many varieties of pumpkins differing in oil content in seeds and its composition. The aim of this study was analysis and comparing fatty acids composition, tocopherols and oil content in seeds of 31 pumpkin varieties. Oils were extracted from the pumpkin seeds, which belong under one of species: Cucurbita maxima, Cucurbita moschata, Cucurbita pepo or Cucurbita pepo var. turbinata. The highest content of oil was found in Green Hubbard (32.69%) variety. The highest content of PUFA and MUFA achieved for Table Queen variety (79.09%) and for Buttercup variety (29.73%), respectively. The predominant fatty acid was linoleic acid (49.63–78.82%), following by oleic acid (8.55–29.73%). The n6:n3 ratio ranged from 59.91 (Yuxijiangbinggua variety) to 607.93 (Cream of the Crop variety). The total tocopherols content was between 0.65 (for Yuxijiangbinggua) and 41.06 mg 100 g−1 (for Snowball). γ-tocopherol was a predominant fraction in all varieties with the highest content on the level 37.52 mg 100 g−1 (Snowball). Potential application areas of examined pumpkin seed oil are consumption, cooking, producing functional food, medicines, but also biolubricants and biofuel. It is assumed a diversity of content and composition of examined pumpkin seed oil was the result of genetic factors.