| Summary: | Cotton is an important economic crop affected by different abiotic stresses at different developmental stages. Salinity limits the growth and productivity of crops worldwide. Na<sup>+</sup>/H<sup>+</sup> antiporters play a key role during the plant development and in its tolerance to salt stress. The aim of the present study was a genome-wide characterization and expression pattern analysis under the salinity stress of the sodium-proton antiporter (NHX) of <i>Gossypium barbadense</i> in comparison with <i>Gossypium hirsutum</i>. In <i>G. barbadense</i>, 25 <i>NHX</i> genes were identified on the basis of the Na<sup>+</sup>_H<sup>+</sup> exchanger domain. All except one of the <i>G. barbadense</i> <i>NHX</i> transporters have an Amiloride motif that is a known inhibitor of Na<sup>+</sup> ions in plants. A phylogenetic analysis inferred three classes of <i>GbNHX</i> genes—viz., Vac (<i>GbNHX1</i>, <i>2</i> and <i>4</i>), Endo (<i>GbNHX6</i>), and PM (<i>GbNHX7</i>). A high number of the stress-related <i>cis</i>-acting elements observed in promoters show their role in tolerance against abiotic stresses. The Ka/Ks values show that the majority of <i>GbNHX</i> genes are subjected to strong purifying selection under the course of evolution. To study the functional divergence of <i>G. barbadense</i> <i>NHX</i> transporters, the real-time gene expression was analyzed under salt stress in the root, stem, and leaf tissues. In <i>G. barbadense</i>, the expression was higher in the stem, while in <i>G. hirsutum</i> the leaf and root showed a high expression. Moreover, our results revealed that <i>NHX2</i> homologues in both species have a high expression under salinity stress at higher time intervals, followed by <i>NHX7</i>. The protein-protein prediction study revealed that <i>GbNHX7</i> is involved in the CBL-CIPK protein interaction pathway. Our study also provided valuable information explaining the molecular mechanism of Na<sup>+</sup> transport for the further functional study of Gossypium <i>NHX</i> genes.
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