Peanut breeders are constantly in search of new sources of genes that confer tolerance or resistance to biotic and abiotic stresses to improve the production and quality. The objective of this study was to evaluate peanut lines generated from interspecific crosses for amounts of wild species introgression, including genes for resistance to peanut root-knot nematodes, tomato spotted wilt virus and leaf spot diseases. Nine diploid Arachis species were crossed with peanut breeding lines and 130 different interspecific hybrid lines were developed. These lines were evaluated for the amount of introgression using RFLP analyses, plant morphology, and disease resistant phenotypes. Based on RFLPs, 41 lines showed measurable introgression and 12 hexaploid-derived lines were polymorphic for at least four probes. Greenhouse and field evaluations indicated that resistance was not present in the lines tested for tomato spotted wilt virus, early leaf spot, or Cylindrocladium black rot. However, resistance approaching that of the wild species was found for the peanut root-knot nematode (Meloidogyne arenaria) among lines derived from crosses with Arachis diogoi, A. correntina, A. batizocoi, and A. cardenasii. Introgression lines were resistant (disease ratings of 1.5 to 4.5 and lesion numbers 8 to 63) compared to Southern Runner (ratings of 5.5 to 6 and lesion numbers of nearly 500) for late leaf spot (Cercosporidium personation) in field evaluations performed in Gainesville, FL over 2 yr. The greatest resistance was found among lines from crosses with A. batizocoi, A. duranensis, A. stenosperma, A. magma, and A. diogoi. Results indicate that it should be possible to identify molecular markers to tag resistance genes for use in conventional breeding programs and stack these genes in highly productive peanut cultivars.