Knowledge of lipid content and composition in the brewing process enables the quality control of the final product. Lipids have a beneficial effect on yeast growth during fermentation as well as deleterious effects on end-product quality. The lipid content of a beer affects its ability to form a stable head of foam and plays an important role in beer staling. Lipid oxidation during wort production is of great interest because of its effect on beer quality: both lipids and their oxidation products are known to have adverse effects on beer flavor, whereas interactions between lipids and protein films stabilizing the gas bubbles are thought to cause the collapse of foam. In this background, the aim of this research was the characterization of the lipid content during a brewing process for evaluating the influence of both technological steps and yeast biomass in the lipid composition of beer. Lipid contents and their fatty acid profile were evaluated in brewing raw materials, wort, and beer. A high-resolution gas chromatography-flame ionization detector (HRGC-FID) system was used for fatty acid determination in lipid extracts. The results of the present study highlighted that the main technological steps influencing the lipid content in brewing byproduct and beer were clarification in a whirlpool and filtration. Moreover, the presence of metabolically active yeast cells (used as starter culture) were found to have a great influence on the fatty acids composition of lipids.