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Abstract Nucleotide-binding site (NBS) domain genes are one of the superfamily of resistance genes involved in plant responses to pathogens. The current study identified 12,820 NBS-domain-containing genes across 34 species covering from mosses to monocots and dicots. These identified genes are classified into 168 classes with several novel domain architecture patterns encompassing significant diversity among plant species. Several classical (NBS, NBS-LRR, TIR-NBS, TIR-NBS-LRR, etc.) and species-specific structural patterns (TIR-NBS-TIR-Cupin_1-Cupin_1, TIR-NBS-Prenyltransf, Sugar_tr-NBSetc.) were discovered. We observed 603 orthogroups (OGs) with some core (most common orthogroups; OG₀, OG₁, OG_2,etc.) and unique (highly specific to species; OG₈₀, OG_82,etc.) OGs with tandem duplications. The expression profiling presented the putative upregulation of OG₂, OG_6,and OG₁₅in different tissues under various biotic and abiotic stresses in susceptible and tolerant plants to cotton leaf curl disease (CLCuD). The genetic variation between susceptible (Coker 312) and tolerant (Mac7)Gossypium hirsutumaccessions identified several unique variants inNBSgenes of Mac7 (6583 variants) and Coker312 (5173 variants). The protein–ligand and proteins-protein interaction showed a strong interaction of some putativeNBSproteins with ADP/ATP and different core proteins of the cotton leaf curl disease virus. The silencing ofGaNBS(OG₂) in resistant cotton through virus-induced gene silencing (VIGS) demonstrated its putative role in virus tittering. The presented study will be further helpful in understanding the plant adaptation mechanism.