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High oleic peanuts and derived food products offer longer shelf life benefits to the food processing industry in addition to multiple health benefits to the consumers. The two mutant alleles, ahFAD2A and ahFAD2B control composition of oleic, linoleic and palmitic acid content in peanut. A total of 563 peanut pre-breeding lines were tested for the presence ahFAD2A and ahFAD2B mutant alleles using allele specific markers. The ahFAD2A mutant allele was present in 82 lines, while none of these lines had ahFAD2B mutant allele. Among botanical types, ahFAD2A mutant allele was more frequent in lines with Virginia growth habit than Spanish bunch although no correlation of ahFAD2A mutant allele with high oleic acid content and growth habit could be established. Oleic and linoleic acid content in 82 pre-breeding lines ranged from 39.70 to 62.70% and 17.76 to 31.95%, respectively, with maximum oleic to linoleic acid ratio of 4. Oleic acid was found to be negatively correlated with linoleic and palmitic acid. Further, pre-breeding lines with ahFAD2A mutant allele, high oleic content and high oleic to linoleic ratio were investigated and novel lines were identified for resistance to late leaf spot, short duration, higher pod yield and other yield related traits. These novel pre-breeding lines can be used as a potential donor in peanut improvement programme and to diversify the primary gene pool including initiating further research on induction of fresh ahFAD2B mutant allele.