Half smooth tongue sole () is a large bottom-dwelling fish that lives in warm water, mainly distributed along the coast of the Bohai Sea and Yellow Sea in China. The half smooth tongue sole is large, grows fast, has a favorable flesh quality and high economic value, and is appreciated by consumers. EPA and DHA are ω-3 polyunsaturated fatty acids that are appreciated for their nutritional and human health values. Pregnant and breastfeeding woman require ω-3 polyunsaturated fatty acids in their diet to promote their baby's development. EPA and DHA lower the incidence of type II diabetes mellitus, hypertension, atherosclerosis and inflammation. Although alphalinolenic acid may be used as a raw material to synthesize EPA and DHA, increasing age and pathological factors mean that the amount of synthesized ω-3 polyunsaturated fatty acids may be insufficient for some tissues and organs, and must be obtained from food intake. Natural aquatic products are very important sources of EPA and DHA. Therefore, it is necessary to determine the EPA and DHA contents to improve farmed fish quality. To study the composition and content of fatty acids in muscles of different half smooth tongue sole families and bred strains, 164 samples from 15 different half smooth tongue sole families were cultivated in the same environment. The muscle fatty acid composition and content of the 164 samples were tested using a gas chromatograph and analyzed comparatively to identify families with high contents of EPA, DHA and EPA+DHA, and to study the relationship between gender and the contents of EPA, DHA and EPA+DHA. There were no significant differences in patterns and types of fatty acids among the different families, while there were significant differences in fatty acid contents. The fatty acid content characteristic of all families was the same:PUFA (polyunsaturated fatty acids)> SFA (saturated acids)> MUFA (monounsaturated fatty acids). The predominant PUFAs were ω-3 polyunsaturated fatty acids. The ratio of ω-3/ω-6 ranged from 4.16 to 5.03. The contents of EPA and DHA were all significantly correlated among families and three high-content families, F13012, F13023 and F13031, were identified. There were no significant correlations between EPA and the gender of the fish; however, there were significant correlations between the gender of the fish and the contents of DHA and EPA+DHA. The contents of DHA and EPA+DHA in males were significantly higher than in females, which will be important for selecting and improving cultivated varieties.