Exploring the phenotypic trait variation and diversity of Lycium ruthenicum germplasm resources can support selection, breeding, and genetic improvement, enhancing agricultural production. This study collected 213 wild Lycium ruthenicum seedlings from a resource nursery in Alxa League, Inner Mongolia. These seedlings originated from eight sources across four provinces. Using 11 pseudo-qualitative traits and 20 quantitative traits, the phenotypic variation of the germplasm was analyzed. The analysis involved the coefficient of variation, Shannon–Wiener index (H), Simpson’s genetic diversity index (D), principal component analysis, correlation analysis, and Q-type cluster analysis. The results showed that the variation range of 31 phenotypic traits across the 213 Lycium ruthenicum germplasm resources was 17.26% to 105.41%, with an average coefficient of variation of 39.85%. The H and D indexes ranged from 0.18 to 1.58 and 0.20 to 0.75, respectively. For the 11 pseudo-qualitative traits, the H and D ranges were 0.18 to 1.58 and 0.07 to 0.74, with average values of 0.77 and 0.42. For the quantitative traits, the H and D ranges were 0.54 to 1.49 and 0.25 to 0.75, with average values of 1.21 and 0.63. This indicates that Lycium ruthenicum germplasm resources exhibit significant phenotypic diversity, with quantitative traits showing higher diversity than pseudo-qualitative traits. Principal component analysis revealed that the cumulative variance contribution rate of the first 10 principal components was 74.03%, comprehensively reflecting the information of the 31 traits. Q-type cluster analysis grouped the 213 Lycium ruthenicum germplasm resources into six clusters, each with distinct phenotypic characteristics. This analysis also identified the trait characteristics and breeding value of each cluster. The results of this study provide valuable information on the genetic improvement, conservation, and evaluation of Lycium ruthenicum germplasm resources.

Lycium ruthenicum Murr. (Black goji), a medicinal and economically valuable crop rich in bioactive compounds, remains genomically understudied despite its expanding cultivation. To overcome limitations of traditional markers in genetic diversity analysis and molecular breeding, we employed specific-locus amplified fragment sequencing (SLAF-seq) to develop genome-wide SNP markers and elucidate the genetic structure of 213 L. ruthenicum accessions from natural and cultivated populations in Alxa, China. We identified 827,630 SLAF tags and 33,121 high-quality SNPs uniformly distributed across 12 chromosomes, establishing the first high-density SNP database for this species. Population genetic analyses revealed three distinct genetic clusters with <60% geographic origin consistency, indicating weakened isolation due to anthropogenic germplasm exchange. The Qinghai Nuomuhong population exhibited the highest genetic diversity (Nei’s index = 0.253; Shannon’s index = 0.352), while low overall polymorphism (average PIC = 0.183) likely reflects SNP biallelic limitations and domestication bottlenecks. Notably, SNP-based clustering showed <40% concordance with phenotypic trait clustering (31 traits), underscoring environmental plasticity as a key driver of morphological variation. This study provides the first genome-wide SNP resource for L. ruthenicum, enabling marker-assisted breeding and highlighting the need for standardized germplasm management to mitigate genetic erosion.