Delta(12) fatty acid desaturase gene has been targeted as a logical candidate controlling the high oleate trait in peanut seeds. By RT-PCR method, the full-length cDNAs of Delta(12) fatty acid desaturase gene were isolated from peanut (Arachis hypogaea L.) genotypes with normal and high ratio of oleic to linoleic acid, which were designated AhFAD2B and AhFAD2B', respectively. Sequence alignment of their coding regions revealed that an extra A was inserted at the position +442 bp of AhFAD2B' sequence of high oleic acid genotypes, which resulted in the shift of open reading frame and a truncated protein AhFAD2B', with the loss of one histidine box involved in metal ion complex required for the reduction of oxygen. Analysis of transcript level showed that the expression of Delta(12) fatty acid desaturase gene in high oleic acid genotype was slightly lower than that in normal genotype. The enzyme activity experiment of yeast (Saccharomyces cerevisiae) cell transformed with AhFAD2B or AhFAD2B' proved that only AhFAD2B gene product showed significant Delta(12) fatty acid desaturase activity, but AhFAD2B' gene product did not. These results suggested that the change of AhFAD2B' gene sequence resulted in lower activity or deactivation of Delta(12) fatty acid desaturase in high oleic acid genotype.