In the literature there are many reports on the composition and properties of pumpkin seed oil; however, few is known about the effect of different stages of seed development on various fatty acid profiles in developing seeds. The objective of this study was to provide the changes of various fatty acid accumulations in seed oil obtained from the seeds of three pumpkin varieties belonging to the species Cucurbita maxima and Cucurbita pepo. Unsaturated acids (oleic and linoleic) were dominant in various fatty acids, which constituted 38.9%-49.1% and 29.4%-42.7% of the total fatty acids at seed maturity for three pumpkin varieties, respectively, while other fatty acid concentrations except for palmitic acid all did not reach 10%. Different varieties exhibited greater effect on various fatty acid contents and the total fatty acid contents in the seeds of pumpkin rather than the species. On the whole, palmitic acid profiles of the seed oil in three varieties all followed the fluctuant decrease during all the stages of seed development, but palmitoleic acid and the total fatty acid profiles of the seed oil in three varieties were just the opposite. Stearic, oleic and linoleic acid profiles of the seed oil in three varieties all experienced the third pattern that fluctuated during all the stages of seed development, but no significant differences in these three fatty acid concentrations were found from the beginning to the end. Linolenic acid concentrations of three varieties were on the decline and ultimately close to zero. Myristic and arachidic acid profiles of the seed oil followed different trends in three varieties. Among them, myristic and arachidic acid profiles of the seed oil of Yinhui-1 fluctuated downward all the time until seed maturity, but those of 0238-1 and Jinhui-2 completely changed oppositely.