To study the genetic difference of the high oleic acid peanut cultivars, twenty-five offspring varieties from five high oleic acid parents were investigated by using gene-based and genetic marker-based diversity analysis approaches. The oleic acid content and O/L of 25 peanut varieties were higher than 75% and 10, respectively. By analyzing the full-length sequence of ahFAD genes of these varieties, a nucleotide substitution of G448A was found in FAD2A, while a nucleotide insertion of A at 442 from the translation starting code in FAD2B was identified. Searching for polymorphism in 25 varieties using 698 SSR markers, 140 SSR markers were found to be polymorphic, which produced a total of 379 alleles. The genetic diversity index value ranged from these 25 varieties were distinguishable by using 17 polymorphic SSR markers. The genetic distance ranged from 0.057 to 0.624 with a mean value of 0.451. Eleven groups were revealed when the genetic similar coefficients of 0.745 which was able to distinguish the parents and the offspring varieties. Taken together, the results may become useful in the pedigree analysis of the high oleic acid peanut varieties and the molecular marker assisted selection in peanut breeding.