Peanut seed fatty acid composition is the most important parameter to determine the oil quality. 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. Overall, these results demonstrate the combined power of genetic analysis with biochemical analysis on peanut fatty acid research.