The hybrid variety of Cucurbita maxima is commercially available and frequently used as food in Bangladesh compared to the native variety. Although the seeds of Cucurbita maxima have nutritional and therapeutic values, people in Bangladesh typically throw them away as waste. If we can explore their physicochemical and biochemical properties, this will add new knowledge to nutrition and food science. Thus, in this study, we compare the physicochemical and biochemical properties of native and hybrid varieties of pumpkin seed and seed oil. Solvent extraction methods were used to obtain oil. The physicochemical properties of the extracted seed oils were examined by titration methods and proximate compositions of pumpkin seeds were determined using the standard method. Tocopherols were analyzed by HPLC and fatty acids were identified by GC/MS as N-acylpyrolidines. The specific gravity, refractive index, viscosity, acid value, saponification value, iodine value and ester values were determined and among them iodine values and acid values were significant (p < 0.05) at native but saponification and ester values were very highly significantly (p < 0.001) at hybrid. The comparative lipid content in the seed of native variety was higher than hybrid (p < 0.01) and protein contents were higher but not significant. However, crude fiber content was higher in the hybrid (p < 0.01). There was no significant differences of moisture, ash, sugar and starch contents in between the two varieties. The total tocopherol, contents were very high (p < 0.001) in native. In hybrid, saturated fatty acids -capric acid, myristic acids were more (p < 0.001) but in native unsaturated fatty acid linoleic and linolenic acids were predominant (p < 0.05). Owing to the considerable differences in the physicochemical composition, the fatty acids and other properties, this study may suggest that the both varieties of pumpkin seed oil may be an alternative good source of edible oil.