Peanuts (Arachis hypogaea L.) are an allotetraploid grain legume mainly cultivated by poor farmers in Africa, in degraded soil and with low input systems. Further understanding nodulation genetic mechanisms could be a relevant option to facilitate the improvement of yield and lift up soil without synthetic fertilizers. We used a subset of 83 chromosome segment substitution lines (CSSLs) derived from the cross between a wild synthetic tetraploid AiAd (Arachis ipaensis × Arachis duranensis)4× and the cultivated variety Fleur11, and evaluated them for traits related to BNF under shade-house conditions. Three treatments were tested: without nitrogen; with nitrogen; and without nitrogen, but with added0 Bradyrhizobium vignae strain ISRA400. The leaf chlorophyll content and total biomass were used as surrogate traits for BNF. We found significant variations for both traits specially linked to BNF, and four QTLs (quantitative trait loci) were consistently mapped. At all QTLs, the wild alleles decreased the value of the trait, indicating a negative effect on BNF. A detailed characterization of the lines carrying those QTLs in controlled conditions showed that the QTLs affected the nitrogen fixation efficiency, nodule colonization, and development. Our results provide new insights into peanut nodulation mechanisms and could be used to target BNF traits in peanut breeding programs.