The objective of this study was to compare the fatty acid (FA) composition of intramuscular fat from the LM of 3 divergent breeds of cattle: Angus (AN, n = 9), Brahman (BR, n = 7), and Romosinuano (RM, n = 11). Cattle were blocked by breed and finished 129 d before slaughter in one year and 157 d in the next year. Longissimus muscle samples were collected from each carcass between the 10th and 13th ribs, trimmed of external fat, frozen in liquid nitrogen, homogenized, and used for fat extraction, using a modified Folch procedure. Extracted fat was analyzed for FA by using a GLC system with an HP-88 capillary column. Fatty acid composition was expressed using both a normalized percentage (%) and gravimetric calculation (mg/g of fresh muscle tissue) in relation to degree of saturation, which was determined using a saturation index (ratio of total SFA to total unsaturated FA). Crude fat determination revealed that LM from AN purebred cattle had the greatest amount of intramuscular fat (7.08%; P = 0.001). Although intramuscular fat of LM from RM contained a reduced percentage of total SFA (P = 0.002) compared with AN, it had the greatest percentage of total PUFA (P < 0.001 and P = 0.020). The percentages of total MUFA were similar among the 3 breeds (P = 0.675). The gravimetric calculation, a measure of actual FA concentration, showed significantly greater concentrations of SFA (26.67 mg/g), MUFA (26.50 mg/g), and PUFA (2.37 mg/g) in LM from AN cattle, as compared with LM from BR and RM cattle (P < 0.001). Interestingly, BR purebreds had the least PUFA concentration (1.49 mg/g; P <or= 0.001) in the LM, although their intramuscular fat content was similar to that of RM (P = 0.924). Regardless of breed, the MUFA proportion was always the greatest (47.58%; P <or= 0.005), whereas PUFA was the least contributor to FA composition (1.49 to 2.37 mg/g and 4.36 to 8.78%; P < 0.001). Beef LM fatty acid composition was characterized by palmitic and oleic acids being the most abundant FA (P < 0.001). These results suggested a genetic variation in FA synthesis and deposition among breeds that influenced both marbling and its composition.