The objective was to compare fatty acid composition of beef Longissimus muscle of White Lamphun and 50% Brahman crossbred raised with fresh Pangola grass. The experiment comprised of each 8 equally White Lamphun and Brahman crossbred steers. The average initial weights were 120-140 kg. They were fed ad libitum with Pangola grass which was cut every 40-50 days. The final slaughter weights were of 275-320 kg, Longissimus dorsi muscle was taken for fatty acid analysis. White Lamphun had a greater fatty acid composition of C15:0, C18:0 and C20:5 n-3 (EPA) (P<0.05) when compared to Brahman crossbred. But polyunsaturated fatty acid (PUFA): saturated fatty acid (SFA) ratio was not significantly different. It was also found conjugated linoleic acid (CLA) in beef (P>0.05) of both cattle breeds. Therefore, the promotion grass fed beef could be an alternative for customer who have health concerns and moreover reduce production costs for farmers.