The high oleic (C18:1) phenotype in peanuts has been previously demonstrated to result from a homozygous recessive genotype (ol₁ol₁ol₂ol₂) in two homeologous fatty acid desaturase genes (FAD2A and FAD2B) with two key SNPs. These mutant SNPs, specifically G448A in FAD2A and 442insA in FAD2B, significantly limit the normal function of the desaturase enzyme activity which converts oleic acid into linoleic acid by the addition of a second double bond in the hydrocarbon chain. Previously, a genotyping assay was developed to detect wild type and mutant alleles in FAD2B. A real-time PCR assay has now been developed to detect wild type and mutant alleles (G448A) in FAD2A using either seed or leaf tissue. This assay was demonstrated to be applicable for the detection of homozygous and heterozygous samples. The FAD2A genotyping assay was validated by employing gas chromatography (GC) to determine total fatty acid composition and by genotyping peanut lines that have been well characterized. Overall, development of rapid assays such as real-time PCR which can identify key genotypes associated with important agronomic traits such as oleic acid, will improve breeding efficiency by targeting desirable genotypes at early stages of development.