Winter mustard is not a common crop in the Southeastern United States. With increased interest to biodiesel production, there has been corresponding interest in mustard as biodiesel crop. The objective of this study was to evaluate the effect of N (0, 50, 100, 150 kg N/ha) on productivity, oil content and oil composition of winter mustard grown for two cropping seasons at three locations in Mississippi (Stoneville, and two locations at Verona, Verona upland silt loam, Verona-SL and Verona upland clay, Verona-C). Seed and oil yields at the three locations were significantly affected by the N treatments. In addition, N application rate significantly affected the concentration of oleic acid in Verona-C location, the concentration of linolenic acid in Stoneville, and the concentration of nervonic acid in the two Verona locations. Generally, seed and oil yields increased with N application relative to the unfertilized control. However, seed and oil yields in the 100 and in 150 kg N/ha treatments at the two Verona locations were not significantly different. At the Verona-C location, the concentration of oleic acid was higher in the 50 kg N/ha treatment than in the other treatments, while the concentration of nervonic acid was higher in the 100 or 150 kg N/ha treatment relative to the other treatments. At the Verona-SL location, nervonic acid concentration was higher in the 0 and 150 kg N/ha treatments than in the 50 kg N/ha treatment. At Stoneville, linolenic acid concentration was higher in the 150 kg N/ha and lower in the 100 kg/N/ha treatment, while it was not different in the other treatments. Overall, the yield of FA palmitic, palmitoleic, stearic, oleic, linoleic, linelenic, arachidic, eicosanoic, behenic, erucic, lignoceric, and nervonic acid increased with higher N rates (100 or 150 kg N/h). Greatest yield of FA in the two Verona locations were achieved in the 100 kg N/ha, while greatest yield of FA at Stoneville was achieved in the highest N rate (150 kg N/ha). This clearly demonstrates that there were other factors than N application rates influencing the overall yields of FA. Winter mustard could be a viable oilseed for the Southeastern US.