Through a comprehensive regional systematic collection, we conducted a genetic diversity analysis of wild red-fruited Lycium resources across the entire northwest region of China. This study provides a valuable genetic basis for germplasm exploration and the selection of new Lycium varieties. Utilizing fluorescence capillary electrophoresis, we carefully screened 16 pairs of SSR primers exhibiting high polymorphism. Subsequently, we inferred the genetic diversity of Lycium germplasm through structure clustering, UPGMA analysis, and molecular AMOVA. The 113 Lycium barbarum samples collected from northwest China exhibited distinct subgroups, namely the Qinghai–Gansu–Ningxia subgroup and the Xinjiang subgroup. These subgroups were clearly distinguishable based on genetic clustering. The genetic diversity within the samples was remarkably rich, as indicated by a mean I value of 1.04, He value of 0.57, and PIC value of 0.73. Notably, the majority of genetic diversity (72.99%) was found within populations, signifying substantial intrapopulation variation. Furthermore, our findings revealed significant genetic differentiation among populations, with a substantial Fst value of 0.27 and gene flow Nm estimated at 0.68. This suggests that the genetic variation levels in northwest Lycium were notably high, primarily driven by pronounced genetic differentiation among populations. Nonetheless, it is important to note that genetic diversity predominantly persists within populations. The observed subpopulation structure of Qinghai–Gansu–Ningxia and Xinjiang regions in northwest China can be primarily attributed to geographical isolation. These geographical barriers have played a pivotal role in shaping the genetic differentiation and structure of Lycium populations in the region. Consequently, our study sheds light on the complex genetic landscape of Northwest Lycium and highlights the significance of considering both within-population diversity and population differentiation in conservation and breeding programs.