Introduction: Studies on the development and validation of semi-quantitative food frequency questionnaires (SQ-FFQ) for assessing omega-3 (Ω-3) long-chain polyunsaturated fatty acids (LC-PUFAs) intake by pregnant women are few. This study aimed to determine the validity of a newly developed SQ-FFQ for assessing the LC-PUFA intake among Indonesian urban pregnant women. Methods: A cross- sectional study was carried out in 2015 on 100 Indonesian pregnant women who were in their late 3rd trimester, living in the urban setting of Jakarta. As a test tool, the SQ-FFQ was administered before the trained nutritionists executed the reference tool of non-consecutive two-day 24-hour dietary recalls (2DRs). The nutrients of interest were a total of Ω-3, eicosapentanoic acid (EPA), docosahexaenoic acid (DHA), alpha-linolenic acid (ALA), total Ω-6, linoleic acid (LA), arachidonic acid (AA), and LC-PUFAs. Statistical correlation, cross-classification and the Bland-Altman plot analysis were done to determine the agreement between tools. Results: Energy-adjusted correlation coefficients between SQ-FFQ and 2DRs were 0.385, 0.349, 0.352, 0.380, 0.338, 0.408, 0.409, 0.331, 0.341 and 0.341 for fat, total Ω-3, ALA, EPA, DHA, total Ω-6, LA, AA and LC-PUFAs, respectively and were statistically significant (p<0.05). Misclassification of these nutrients from SQ-FFQ and 2DRs was <6%. The Bland-Altman plots showed most of the points fell within the 95% limits of acceptable agreement for DHA, EPA, and LA. Conclusion: The newly developed SQ-FFQ of this study is a valid instrument for assessing of Ω-3 LC-PUFAs intake among Indonesian pregnant women living in urban area. Its further validation with relevant biomarkers is recommended.