Abstract Background To assess severity and risk of an essential fatty acid deficiency in children <2 years with parenteral nutrition–associated cholestasis on long‐term 100% fish‐oil lipid injectable emulsion. Methods This longitudinal descriptive cohort study included patients receiving fish‐oil lipid injectable emulsion (1 g/kg/day). Triene: tetraene ratios were monitored for up to 4 years and classified as mildly elevated (≥0.05 and <0.2) or essential fatty acid deficiency (≥0.2). Results One hundred and twenty‐seven patients with a baseline median age of 14 weeks were included. Serum docosahexaenoic acid and eicosapentaenoic acid levels markedly increased, whereas arachidonic acid, linoleic acid, and α‐linolenic acid levels decreased before stabilizing. Median triene: tetraene ratios peaked at 0.027 at week 8 and then stabilized within a range of 0.015 and 0.020 from week 16 until the end of the study. Seven patients had mildly elevated triene: tetraene ratio at the end of the study. Three infants had an essential fatty acid deficiency, but none demonstrated clinical signs consistent with this deficiency. One deficiency was attributed to a laboratory error; two were associated with adverse events. All patients resolved with the continuation of fish‐oil lipid injectable emulsion. Conclusion Children with parenteral nutrition–associated cholestasis on long‐term fish‐oil lipid injectable emulsion are at low risk for a clinical or biochemical essential fatty acid deficiency. These findings indicate that despite its low content of linoleic acid and α‐linolenic acid, long‐term 1 g/kg/day of 100% fish‐oil lipid injectable emulsion is not associated with an essential fatty acid deficiency.