The objective of this investigation was to provide a comprehensive analysis of the total lipid composition of present-day retail beef meat available at the consumer level and to evaluate the total lipid composition with special emphasis on the nutritional value. For this purpose, 40 beef cuts were obtained from four cattle farms based on either a natural grazing system (NGS) or an intensive production system (IPS). The total lipid composition was analyzed using complementary chemical and chromatographic procedures. The content of n-3 LC-PUFA, CLA, total trans-18:1, and branched-chain fatty acids was significantly higher in NGS beef than in IPS beef. The trans-18:1 and CLA profiles were affected by the different production systems, whereby they can be utilized empirically to differentiate between feeding regimen and production management. Fatty acid ratios that have health implications ( n-6/ n-3, LA/alphaLNA, and AA/EPA) were remarkably beneficial for NGS beef compared with IPS beef. In conclusion, from the human health perspective, beef raised on NGS is clearly superior with regard to a more favorable fatty acid profile in comparison to IPS beef.