A high-oleate trait in the cultivated peanut ( Arachis hypogaea L.) was reported to rely on the allelic composition of the two homeologous, microsomal oleoyl-PC desaturase genes ( ahFAD2A or ahFAD2B). The enzyme activity of either ahFAD2A or ahFAD2B is sufficient for a normal oleate phenotype, and a significant reduction in the levels of ahFAD2B and a mutation in ahFAD2A were reported to be responsible for the high-oleate phenotype in one chemically induced mutant (M2-225) and one derived from a naturally occurring (8-2122) mutant. Here, we report an insertion of the same miniature inverted-repeat transposable element (MITE) in the ahFAD2B gene in another chemically induced mutant (Mycogen-Flavo) and the previously characterized M2-225 mutant. In both cases, this MITE insertion in ahFAD2B causes a frameshift, resulting in a putatively truncated protein sequence in both mutants. The insertion of this MITE in ahFAD2B, in addition to the point mutation in ahFAD2A, appears to be the cause of the high-oleate phenotype in Mycogen-Flavo and M2-225 mutants. Utilizing sequences of the MITE, we developed a DNA marker strategy to differentiate the two insertion-containing mutants from the normal oleate peanut variety (AT-108) and the naturally occurring, high-oleate mutant 8-2122. Reverse transcript-PCR/differential digestion results reveal the expression of both ahFAD2A and ahFAD2B genes in Mycogen-Flavo mutant. This result is in contrast to the observation that ahFAD2B transcripts are greatly reduced in the M2-225 mutant having the MITE insertion further 3' in ahFAD2B gene.