Plant MAP3Ks have expanded significantly compared to their metazoan counterparts. A new, sequential workflow combining multispecies ortholog clustering and newly built, family-specific HMMs is used to identify the MAP3K gene family within seven plant species, allowing for a refinement of previously proposed gene family cladding and the novel identification of the MAP3K gene families in the allotetraploid cotton Gossypium hirsutum and newly sequenced monocot seagrass Zostera marina. The MAP3K gene family architecture is further refined and validated using bioinformatics analyses before the recently characterized Arabidopsis Raf-like MAP3K ILK1 is identified and characterized in upland cotton. Transient gene silencing reveals an increase in RKN susceptibility following GhILK1.1 silencing in the susceptible TM1 cultivar. No changes in susceptibility were seen in the resistant M240 cultivar or against reniform nematodes. GhILK1.1 is only the second cotton gene characterized to have a direct role in mediating RKN resistance.