Saussurea involucrata is a rare perennial herb endemic to the alpine zone of the Tianshan Mountains, possessing significant medicinal value yet facing severe threats from overharvesting and habitat fragmentation. The core distribution area and recognized genetic differentiation center of this species are located in the Bayinbuluke region of the Western Tianshan Mountains. In contrast, the genetic distinctiveness and conservation status of populations in the Eastern Tianshan Mountains have remained unclear. To clarify the genetic relationships, we conducted an integrated analysis using nuclear microsatellite (SSR) markers as well as chloroplast (cpDNA) and nuclear ribosomal DNA (nrDNA) sequences on 16 populations (5 from the Eastern Tianshan Mountains and 11 from the Bayinbuluke region). The results showed that the Eastern Tianshan Mountains populations exhibited higher genetic diversity (mean He = 0.5568). Bayesian clustering and principal coordinate analysis (PCoA) clearly separated all populations into two genetic groups corresponding to the two geographical regions. Notably, private haplotypes (cpDNA H1 and nrDNA H7) were identified exclusively in the Eastern Tianshan populations, and no recent genetic bottleneck was detected, indicating historical demographic stability. These findings demonstrate significant genetic differentiation and a unique evolutionary trajectory in the Eastern Tianshan Mountains populations, likely resulting from long-term geographical isolation and local adaptation to arid environments. Therefore, we propose that these populations be managed as an independent Management Unit (MU) to preserve their unique genetic legacy. This study provides critical genetic evidence for refining conservation strategies and promoting the sustainable use of this endangered species, while the established molecular marker system also offers a reliable framework for its geographical traceability.