This study aims to investigate associations between omega-3 polyunsaturated fatty acids (PUFAs) and myopia. Two-sample Mendelian randomization (MR) was conducted to estimate the associations between plasma levels of omega-3 PUFAs and three traits of myopia, including myopia, high myopia (HM), and refractive spherical equivalent (RSE). Summary data-based Mendelian randomization (SMR) and colocalization analysis were conducted to examine the associations between the FADS1 and FADS2 genes and three traits of myopia in European populations. The cross-sectional study based on the Korean National Health and Nutrition Examination Survey (KNHANES) was performed to explore the relationship in East Asian adolescents. In the Two-sample MR study, plasma levels of total omega-3 PUFAs (0.993[0.990, 0.996]), Docosahexaenoic acid (DHA) (0.992[0.989, 0.996]), and Eicosapentaenoic Acid (EPA) (0.969[0.955, 0.983]) were found to be significantly and inversely associated with myopia in European populations, and similar results were shown in HM and RSE. SMR ( = -0.028,  < 0.05; HEIDI test > 0.05) and colocalization analysis (PPH4 = 0.926) identified an association between the expression of the FADS1 gene in the retina, crucial in PUFAs biosynthesis, and high myopia. In the cross-sectional study, daily intake of DHA and EPA was found to be significantly associated with HM and RSE in East Asian adolescents. This study suggests a potential link between elevated omega-3 PUFAs levels and a reduced risk of myopia, highlighting the involvement of the PUFAs biosynthesis pathway in HM among European populations. Further exploration is needed to uncover the underlying processes of this causal association.