Onchidiidae which is widely distributed in China and rich in resources is one kind of economic gastropoda with nutritional value, despite reports for molecular biology, ecology, developmental, physiological and ecological breeding field of research. The analysis of biochemical components of Onchidiidae is conductive to its development and utilization of resources. In view of this, this study determined and compared four different species of Onchidiidae (Peronia verruculata, Onchidium struma, Platevindex mortoni and Paraoncidium reevesii) of total fat (TF) content, lipid and fatty acid composition. The experiments were conducted to investigate total lipid(TL) class and fatty acid composition of Onchidiidae by GC-MS and others. Results show that: ① Four different onchidiids muscle total fat are mainly composed of phospholipids (PL), free fatty acid (FFA), cholesterol (Cho) and triglyceride (TG); the PL/TL content of P. verruculata’s muscle tissue is as high as 73.12%, significantly higher than the other three species (P<0.05); the PL/TL content of O. struma muscle tissue is only 26.23%, significantly lower than the other three species (P<0.05); the TG/TL content of O. struma muscle tissue amounted to 2.75% of muscle tissue, significantly higher than the other three species (P<0.05); FFA/TL of P. verruculata’s muscle tissue is as high as 19.16%, significantly higher than the other three species (P<0.05). ② The fatty acid composition of muscle tissue of four different onchidiids is more balanced (S:M:P close to 1:1:1). The total saturated fatty acids in muscle tissue (Σ SFA) levels of O. struma and P. verruculata were above 35%, Σ SFA of P. mortoni muscle tissue is only 29.58%, significantly lower than the other three species (P<0.05); the four species of Onchidiidae total monounsaturated fatty acid (Σ MUFA) content were similar; the total polyunsaturated fatty acid content of P. mortoni’s muscle tissue is 34.25%, significantly higher than the other three species (P<0.05). The C20:4n6 (ARA) of P. mortonian in muscle tissue content and the C22:5n3 (DPA) of P. reevesii’s muscle tissue were significantly higher than that of the other three onchidiids, C20:5n3 (EPA) content in four onchidiids were significantly higher than the C22:6n3 (DHA). Studies have shown that four species contained higher ω-3 polyunsaturated fatty acids, which suggests that onchidiids’ muscle tissue has high lipid nutrition value, in addition, the composition of lipid and fatty acid of all four has a significant difference, may be related to its habitats and species diversity. The purpose of this study was to evaluate the nutritional value of different kinds of Onchidiidae, provide theoretical basis for the development and utilization of its resources, and provide theory support for the ecological culture and enhancement.