Key message <p>The RKN resistance locus <i>Rmi1</i> was fine-mapped to two genes on chromosome 10, a glycosyl hydrolase family 9 β-1,4-endoglucanase gene and a type I pectin methylesterase gene.</p> Abstract <p>Root-knot nematodes (<i>Meloidogyne</i> spp.) are a serious threat to soybean production in the southeast USA, with yield losses of more than $165 million in 2023. Development and deployment of resistant soybean cultivars is the most effective strategy for managing these nematode pests; however, the identity of the resistance genes and underlying mechanism of resistance remains obscure. An additive resistance gene, <i>Resistance to M. incognita-1</i> (<i>Rmi1</i>), to the predominant species, was first identified in soybean&#xa0;cultivar Forrest but never mapped to a genomic region. Multiple mapping studies have identified a major quantitative trait locus (QTL) with additive action on chromosome 10. In this study, a population consisting of 170 F_2:3 families derived from a cross of Bossier (susceptible) × Forrest (resistant) was initially used to confirm that <i>Rmi1</i> is in the chromosome 10 QTL. Subsequently, 884 F_5:6 recombinant inbred lines (RILs) derived from the same cross were used to fine-map the <i>Rmi1</i> causal gene(s) to two genes – a β-1,4-endoglucanase (<i>Glyma.10G017000</i>, <i>EG</i>) and a pectin methylesterase/methylesterase inhibitor (<i>Glyma.10G017100</i>, <i>PME1</i>). Both gene candidates have the potential to play a role in the resistance response to <i>M. incognita.</i> Both gene promoters harbor SNPs and indels and the encoded proteins exhibit amino acid polymorphisms, including a premature stop in <i>PME1</i> of resistant soybeans. Additionally, both genes show a higher expression level in susceptible roots compared to resistant roots in the absence of infection. This suggests that <i>Rmi1</i> may confer one or more pre-existing differences related to cell wall modification in soybean roots, ultimately leading to a decrease in susceptibility.</p>