Wild peanut species are useful genetic resources for improving the levels of disease/pest resistance and for enhancing the quality of seed composition by interspecific hybridization. The variation in oil content and fatty acid composition of wild peanut species in the United States Department of Agriculture germplasm collection is unknown. Seeds available from 39 wild species (plus a cultivated peanut) were requested from the U.S. peanut germplasm collection. Oil content was measured using nuclear magnetic resonance, fatty acid composition was analysed using gas chromatography, and the D150N functional mutation of the FAD2A gene was screened by real-time PCR. Significant variability in oil content (41.7–61.3%) was identified among the wild peanut species. Arachis magna contained significantly more oil (61%) than cultivated peanut (56%). There was no functional mutation identified within the FAD2A gene target, and no wild species were identified with a high ratio of oleic acid to linoleic acid. The results from gas chromatography and real-time PCR analyses were consistent. However, Arachis sylvestris contained a significantly higher amount (22%) of long-chain fatty acid (LCFA) than the cultivated peanut (4%). Thus, A. magna and A. sylvestris may be good breeding materials to use for increasing oil content or LCFA composition of cultivated peanuts in breeding programs.