The emergence of heterosis is a crucial marker of hybrid potential in rapeseed breeding for both hybrid and open-pollinated cultivars. However, testing the combining ability of the parents in the field consumes a great deal of manpower. An efficient way to evaluate heterosis and heritability is to use a line×tester analysis. An experiment was performed in 2022 in the research area of department of Plant Breeding and Genetics, University of Agriculture, Faisalabad. Line×tester analysis was performed with ten lines and three testers for the development of thirty F1 crosses. It was used to estimate heritability and heterosis of various fatty acids related traits as: oil contents, protein contents, glucosinolate contents, and erucic acid content. For this, selected better performer lines (high yield) and poor performer (low-yield) testers from ongoing research were crossed and the data were recorded for different fatty acids-related traits. The recorded data were subjected to Analysis of Variance followed by estimation of heterosis and narrow sense and broad sense heritability. Using three alternative forms of heterosis estimates—heterosis over mid parent, heterosis over better parent, and heterosis over standard check—the thirty F1 hybrids that developed were assessed, along with one check variety (Chakwal-second). Crosses 40980×26240 (for oil), 40979×26240 (for glucosinolate contents), 40978×26240 (for erucic acid contents) had positive and significant heterosis over the commercial hybrids for the studied traits. High narrow sense heritability was observed for erucic acid contents followed by protein contents and glucosinolate contents. The hybrid variety in rapeseed might be developed using the crossings 40978×26240, 40979×26240, and 40980×26240.