| Summary: | Within the family Solanaceae, <i>Withania</i> is a small genus belonging to the Solanoideae subfamily. Here, we report the de novo assembled chloroplast genome sequences of <i>W. coagulans, W. adpressa</i>, and <i>W. riebeckii.</i> The length of these genomes ranged from 154,162 to 154,364 base pairs (bp). These genomes contained a pair of inverted repeats (IRa and IRb) ranging from 25,029 to 25,071 bp that were separated by a large single-copy (LSC) region of 85,635–85,765 bp and a small single-copy (SSC) region of 18,457–18,469 bp. We analyzed the structural organization, gene content and order, guanine-cytosine content, codon usage, RNA-editing sites, microsatellites, oligonucleotide and tandem repeats, and substitutions of <i>Withania</i> plastomes, which revealed high similarities among the species. Comparative analysis among the <i>Withania</i> species also highlighted 10 divergent hotspots that could potentially be used for molecular marker development, phylogenetic analysis, and species identification. Furthermore, our analyses showed that even three mutational hotspots (<i>rps</i>4-<i>trn</i>T, <i>trn</i>M-<i>atp</i>E, and <i>rps</i>15) were sufficient to discriminate the <i>Withania</i> species included in current study.
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