The influence of feeding systems on the levels of functional lipids and other fatty acid concentrations in Australian beef was examined. Rump, strip loin and blade cuts obtained from grass feeding, short-term grain feeding (80 days; STGF) and long-term grain feedlot rations (150-200 days; LTFL) were used in the present study. The typical Australian feedlot ration contains more than 50% barley and/or sorghum and balanced with whole cottonseed and protein meals were used as feed for STGF and LTFL regimens. Meat cuts from 18 cattle for each feeding regimen were trimmed of visible fat and connective tissue and then minced (300 g lean beef); replicate samples of 7 g were used for fatty acid (FA) analysis. There was a significantly higher level of total omega-3 (n-3) and long chain n-3 FA in grass-fed beef (P< 0.0001) than the grain-fed groups regardless of cut types. Cuts from STGF beef had significantly reduced levels of n-3 FA and conjugated linoleic acid (CLA) and similar levels of saturated, monounsaturated and n-6 FA compared with grass feeding (P < 0.001). Cuts from LTFL beef had higher levels of saturated, monounsaturated,n-6 FA and trans 18:1 than similar cuts from the other two groups (P<0.01), indicating that increased length of grain feeding was associated with more fat deposited in the carcass. There was a step-wise increase in trans 18:1 content from grass to STGF to LTGF, suggesting grain feeding elevates trans FA in beef, probably because of increased intake of 18:2n-6. Only grass-fed beef reached the target of more than 30mg of long chain n-3 FA/100 g muscle as recommended by Food Standard Australia and New Zealand for a food to be considered a source of omega-3 fatty acids. The proportions of trans 18:1 and n-6 FA were higher (P<0.001) for both grain-fed beef groups than grass-fed beef. Data from the present study show that grain feeding decreases functional lipid components (long chain n-3 FA and CLA) in Australian beef regardless of meat cuts, while increasing total trans 18:1 and saturated FA levels.