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Root-knot nematode (RKN) infections are among the most serious soil-borne diseases in the world, and tomato is a common host of RKNs. WRKY transcription factors are involved in complex, diverse biological processes in plants. In a previous study, a resistant variety, LA3858 (<i>Mi-3</i>/<i>Mi-3</i>), was treated at different soil temperatures before RNA-seq, and six differentially expressed genes (DEGs) encoding WRKY proteins were screened. In this study, cloning and sequencing were used to identify six target DEGs encoding SlWRKY1, SlWRKY13, SlWRKY30, SlWRKY41, SlWRKY46, and SlWRKY80. Conserved domain identification and phylogenetic tree analysis showed that SlWRKY1, SlWRKY13, and SlWRKY46 have similar functions and are mainly involved in plant growth and development and abiotic stress responses. SlWRKY30 and SlWRKY41 share high homology, while AtWRKY46 and AtWRKY70, which are highly homologous to SlWRKY80, play an important role in the disease resistance of <i>A. thaliana</i>. Considering these findings combined with the high level of <i>SlWRKY80</i> expression observed in the roots and leaves of the resistant variety Motelle (<i>Mi-1</i>/<i>Mi-1</i>) and the continuous upregulation of <i>SlWRKY80</i> expression in the roots after inoculation of Motelle with <i>M. incognita</i>, it is speculated that <i>SlWRKY80</i> plays an important role in the <i>Mi-1</i>-mediated disease resistance pathway. Further study revealed that SlWRKY80 is a typical nuclear-localized protein, and a virus-induced gene silencing (VIGS) assay verified that <i>SlWRKY80</i> is involved in tomato resistance to RKNs as a positive regulator. SA and JA signals play an important role in <i>Mi-1</i>-mediated resistance to RKNs. <i>SlWRKY80</i> was able to respond rapidly to treatment with both plant hormones, which indicated that <i>SlWRKY80</i> might be involved in disease resistance regulation through various immune pathways.