A 16-week feeding trial was conducted to investigate effects of replacing dietary fish oil (FO) with docosahexaenoic acid (DHA)-rich microbial oil (MO) from Schizochytrium sp. (T18) on membrane lipid composition of Atlantic salmon parr liver and muscle tissues. Four nutritionally balanced diets were formulated with varying levels of FO, MO, and canola oil (CO), including a control diet with 20% FO, a secondary control diet with 10% FO and 10% CO, and two experimental diets that completely replaced FO with a low (5%) and high (10%) proportion of MO. No significant differences were observed in growth parameters (81–98 g; weight gain), total lipid class composition, and total sterol content among the dietary treatments. However, there were significant differences in the proportions of individual ꞷ3 and ꞷ6 fatty acids in both liver and muscle tissues, reflecting the different dietary treatments. Notably, the presence of low eicosapentaenoic acid (EPA) in the MO diets did not affect the growth performance of the fish, suggesting a lower requirement for EPA in the diet and a greater necessity for DHA. The results also showed that DHA was present in very high proportions in the cellular membrane, particularly in muscle tissue, with low levels of linoleic acid and alpha-linolenic acid. Overall, the findings suggest that MO derived from Schizochytrium sp. (T18) could be a potential substitute for FO in the diet of farmed Atlantic salmon.