Caragana acanthophylla Kom. is an ecologically important drought-tolerant shrub in Xinjiang, China, but species-specific molecular markers for germplasm characterization remain limited. We sampled 93 individuals from 11 localities representing the currently known distribution of C. acanthophylla in Xinjiang. Three individuals per locality (33 in total) were whole-genome resequenced, yielding 2,873,410 high-quality SNPs and 5,679,915 InDels. Genome-wide SNP-based PCA and genetic relationship analysis provided an independent high-resolution assessment of the 33 resequenced individuals. From 34 candidate primer pairs, eight polymorphic InDel markers with stable amplification and clear genotyping profiles were retained and applied to all 93 individuals. The SNP dataset revealed clear regional differentiation and finer locality-associated relationships. Analysis of the same 33 individuals with the eight InDel loci recovered part of this broad pattern, particularly the differentiation of the western YL materials, but showed lower fine-scale resolution. Across all 93 individuals, the InDel panel revealed moderate to low marker-level genetic diversity and detectable regional differentiation. AMOVA attributed 67.00% of the variation to differences among the 11 original sampling localities, while the five exploratory analytical groups showed a similar among-group component (68.37%). The Mantel correlation detected across all 93 individuals (r = 0.801, p < 0.001) disappeared after YL was excluded (r = −0.032, p = 0.724), indicating that the overall spatial signal was largely driven by the geographic separation of YL. These results support the eight-marker panel as a practical, low-cost tool for preliminary germplasm characterization and broader sample screening, while genome-wide SNP data provide substantially greater resolution for population-level inference.

Calligonum sect. Medusa is an important resource for desert ecological restoration, but practical criteria for elite germplasm screening and reliable methods for identity verification remain limited. We evaluated 13 morphological, physiological, and biochemical traits in 121 individuals conserved ex situ at the Turpan Campus of Yili-Tupan National Botanical Garden (electable) using principal component analysis, membership-function evaluation, hierarchical clustering, and stepwise regression; 20 individuals were further genotyped at 13 simple sequence repeat (SSR) loci. Trait coefficients of variation ranged from 6.54% to 53.79%, and five principal components explained 68.40% of the total variation. Five candidate elite individuals (ZLY37, ZLY40, ZLY44, ZLY38, and ZLY01) were identified. A reduced six-trait model retained crown width, predawn water potential, plant height, soluble sugar content, plant-form integrity, and assimilating-branch length, from which preliminary empirical reference thresholds were derived. Of the 13 SSR loci, 10 were polymorphic (mean PIC = 0.357), and a candidate five-locus panel distinguished the same 20 individuals used for its selection and enabled fingerprint construction for the five candidates. This study provides a practical framework for preliminary elite germplasm screening and molecular identity verification in Calligonum sect. Medusa, supporting desert ecological restoration, germplasm management, and subsequent cultivar development.