This study aimed to compare the fatty acids composition of Brycon cephalus (matrinxã) through four different methods of fatty acid quantification. Samples were collected from Teles Pires River, near from Lucas do Rio Verde, Mato Grosso state, central Amazonia region. Fish samples were divided into three lots, each containing ten fish of similar sizing. Total lipids were extracted by Bligh and Dyer (1959) method. Fatty acid methyl esters were prepared by Hartman and Lago (1973) method. Methyl esters were separated by gas chromatography using a Thermo 3300 gas chromatograph fitted with a flame ionization detector. The methods applied were: area normalization (MAN), internal standard (MIS), alternative theoretical (MAT) and alternative experimental (MAE). A significant difference (P<0.05) was observed between the methods applied. MAN showed no significant difference (P>0.05) in MUFA, PUFA, n-3 and n-6 fatty acids contents. The other three methods of fatty acids quantification (MIS, MAT and MAE) showed a significant difference (P<0.05) between those parameters. This may be due to the characteristics of MAN, which does not supply the real fatty acids content in samples as reported in literature, since the results are expressed as percentages from relative areas, which are difficult to interpret and translate to nutritional label. MIS, MAT and MAE supplied information as mass about fatty acid composition of B. cephalus, which showed great contents of n-3 fatty acids, proximately 68 mg g-1 to MIS and 74 mg g-1 to MAE and MAT. EPA and DHA content totalized 104.37 mg 100g-1. Comparing to the World Health Organization recommendations, of 200 – 500 mg of those fatty acids per day, a serving of 100g of studied species supplied about 50% of daily intake of EPA+DHA. Thus, MIS showed the most accurate results, whereas MAT and MAE, in general, overestimated the results.