As an endemic fish vulnerable to habitat fragmentation and hydrological changes in the arid Shule River Basin, Triplophysa yarkandensis macroptera lacks sufficient genetic tools for effective conservation monitoring. To address this knowledge gap, we report the first development of polymorphic simple sequence repeat (SSR) markers for this subspecies via Restriction site-associated DNA sequencing (RAD-seq), and assess the genetic diversity and population structure of three geographical populations (Yumen, Guazhou, and Qiaowan). From 54 candidate loci that met specific criteria, we successfully developed 17 novel, highly polymorphic SSR markers. Genetic diversity varied among populations with Na ranging from 2 to 19 (highest at locus SSRQ26 in the Qiaowan population), Ne from 1.385 to 11.560, and no null alleles detected. The Qiaowan population exhibited the highest allele count. Furthermore, population structure analysis revealed no significant genetic differentiation between the Yumen and Guazhou populations, whereas the Qiaowan population was genetically distinct. The newly identified SSR markers and the baseline genetic data provide a powerful toolkit for ongoing genetic monitoring and the designation of distinct Management Units (MUs), which is of critical conservation importance for preserving this endemic species within the unique and highly fragmented ecological context of the arid Shule River Basin in northwestern China.