To better estimate diversity within the cultivated peanut, germplasm representing 33 South American peanut accessions from six countries was evaluated for isozyme polymorphisms. Only three of 18 isozymes—glutamate oxaloacetate transaminase (GOT), isocitrate dehydrogenase (IDH), and phosphohexose isomerase (PHI) — were consistently variant, each displaying two banding patterns. The variant banding patterns were observed in 18, 9, and 9% of the genotypes for GOT, IDH, and PHI, respectively. Isozyme variation in A. hypogaea could not be associated with subspecies or botanical variety. Thirty interspecific hybrids and their parents were also evaluated for isozyme polymorphisms. Flower tissues showed variations for the following isozymes: alanine aminopeptidase (AAP), arginine aminopeptidase (AMP), glutamate oxaloacetate transaminase (GOT), malate dehydrogenase (MDH), and phosphohexose isomerase (PHI). A specific PHI band pattern was observed in all three hybrid lines with early leafspot resistance, as well as three of the four lines associated with high yield. For seed tissue, the absence of a fast-moving leucine aminopeptidase (LAP) band was associated with three of the four high-yielding lines. A comparison of flower and seed isozyme banding patterns revealed that the banding pattern was different for GOT, IDH, LAP, MDH, and PHI. IDH and MDH were variant in seeds but not flowers, and GOT was more polymorphic in flowers than seeds. The investigation indicates that isozymes may serve as molecular markers for interspecific hybrid identification and gene introgression to the A. hypogaea genome, and possibly for identifying lines with useful resistances.