Plant oils rich in oleate are considered superior products compared to oils rich in polyunsaturated fatty acids. Peanut (Arachis hypogaea L.) is one of the major oilseed crops, and high oleate mutant varieties with as much as 85% oleate have been reported. We examined the possibility that this mutant phenotype resulted from reduction in the activity or the transcript level of microsomal oleoyl-PC desaturase. Two independently generated high oleate mutants, M2-225 and 8-2122, and their partially isogenic lines with a normal oleate phenotype were used in this study. Two cDNA sequences coding for microsomal oleoyl-PC desaturases, ahFAD2A and ahFAD2B, have been isolated from the developing peanut seed with a normal oleate phenotype. Cultivated peanut is an allotetraploid, and sequence comparisons with the genes from the putative diploid progenitor species suggested that ahFAD2A and ahFAD2B are non-allelic, but homeologous genes originating from two different diploid species. Northern analysis showed that the transcripts of oleoyl-PC desaturases are highly abundant in both normal and high oleate peanut seeds in the second stage of development. Differential digestion of the RT-PCR products revealed a restriction site polymorphism between ahFAD2A and ahFAD2B, and allowed us to examine the level of transcript expressed from each gene. The results indicate that ahFAD2A is expressed in both normal and high oleate peanut seeds, but the steady state level of the ahFAD2B transcript is severely reduced in the high oleate peanut varieties. These data suggested that the reduction in ahFAD2B transcript level in the developing seeds is correlated with the high oleate trait.