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Lagerstroemia indica L. is a well-known ornamental plant with large pyramidal racemes, long flower duration, and diverse colors and cultivars. It has been cultivated for nearly 1600 years and is essential for investigating the germplasm and assessing genetic variation to support international cultivar identification and breeding programs. In this study, 20 common Lagerstroemia indica cultivars from different varietal groups and flower morphologies, as well as multiple wild relative species, were analyzed to investigate the maternal donor of Lagerstroemia indica cultivars and to discover the genetic variation and relationships among cultivars based on plastome and nuclear ribosomal DNA (nrDNA) sequences. A total of 47 single nucleotide polymorphisms (SNPs) and 24 insertion/deletions (indels) were identified in the 20 L. indica cultivars' plastome and 25 SNPs were identified in the nrDNA. Phylogenetic analysis based on the plastome sequences showed that all the cultivars formed a clade with the species of L. indica, indicating that L. indica was the maternal donor of the cultivars. Population structure and PCA analyses supported two clades of cultivars, which exhibited significant genetic differences according to the plastome dataset. The results of the nrDNA supported that all 20 cultivars were divided into three clades and most of the cultivars had at least two genetic backgrounds and higher gene flow. Our results suggest that the plastome and nrDNA sequences can be used as molecular markers for assessing the genetic variation and relationships of L. indica cultivars. [ABSTRACT FROM AUTHOR]