Abstract MicroRNAs (miRNAs) play important roles in regulating salt tolerance in Dongxiang wild rice (DXWR, Oryza rufipogon Griff.). The development of salt-responsive miRNA-simple sequence repeat (SSR) markers will significantly bolster research on DXWR, providing novel tools for exploring salt-tolerant genetic resources and advancing the development of salt-tolerant rice varieties. In the present study, a total of 137 miRNA-SSR markers were successfully developed, specifically derived from miRNAs responsive to salt stress in DXWR. Subsequently, a subset of 20 markers was randomly selected for validation across three distinct DXWR populations, along with 35 modern rice varieties. Notably, 13 of these markers exhibited remarkable polymorphism. The polymorphic markers collectively amplified 52 SSR loci, averaging four alleles per locus. The polymorphism information content values associated with these loci spanned from 0.23 to 0.70, with a mean value of 0.49. Particularly noteworthy is the miR162a-SSR marker, which demonstrated distinct allelic patterns and holds potential as a diagnostic marker for discriminating the salt-tolerant rice varieties from the non-tolerant varieties. This study provides a valuable tool for genetic analysis and precision breeding, facilitating the identification and utilization of valuable salt-tolerant genetic resources.