Technical Abstract: Fatty acid desaturase 2 (FAD2) is one of the key enzymes for the conversion of oleic acid to linoleic acid in plants. Two homoeologous genes (FAD2A on the A genome and FAD2B on the B genome) have been characterized in cultivated peanut (Arachis hypogaea L.). However, the contribution from each homoeologous gene to enhance the amount of oleic acid and to alter fatty acid profiles is not clear. A panel of 55 peanut lines was analyzed for fatty acid composition with gas chromatography (GC) and also genotyped with SNP markers from the FAD2 genes by real-time PCR. Significant variation in fatty acid composition was identified and the ratio of oleic acid to linoleic acid (O/L) ranged from 1.23 to 56.45. In terms of the FAD2 gene mutation, the assayed lines were classified into four genotypes: wild type (Ol1Ol1Ol2Ol2), single functional homozygous mutation on the A genome (ol1ol1Ol2Ol2), single functional homozygous mutation on the B genome (Ol1Ol1ol2ol2), and a double mutation on both A and B genomes (ol1ol1ol2ol2). Each genotype has a significantly different fatty acid profile. Both FAD2A and FAD2B are involved in the conversion of oleic acid to linoleic acid in peanut. Each gene mutation can increase the O/L ratio; but, the double mutation has a highly significant affect on increasing the O/L ratio. The mutant genotype (ol) also has a significant effect on increasing the amount of gadoleic acid and decreasing the amount of palmitic acid. Overall, these results demonstrate the combined power of genetic analysis with biochemical analysis on peanut fatty acid research.