Murray cod (Maccullochella peelii), an emerging species in Chinese aquaculture, shows considerable potential for commercial cultivation. To support genetic resource management and facilitate selective breeding programs, we conducted the first characterization of microsatellite (SSR) loci from the Murray cod genome using restriction-site associated DNA sequencing (RAD-seq) and developed highly polymorphic SSR markers. The results showed that a total of 3 598 complete microsatellite loci were identified from the reduced-epresentation genome of Murray cod. Among these, dinucleotide repeats were the most abundant (2 018 loci, 56.09%), with AC and TG as the predominant motifs (21.41% and 21.01%, respectively). Trinucleotide repeats were the second most frequent type (1 169 loci, 32.49%), among which TTA was the dominant motif (6.33%). Tetranucleotide and pentanucleotide repeats were less common, representing 9.20% and 2.00% of the total SSRs, respectively, with TTTA and TTAGG as the predominant motifs (4.53% and 19.15%, respectively). Hexanucleotide repeats were the least abundant, accounting for only 0.22%, and no dominant motifs were identified. Furthermore, 19 highly polymorphic SSR markers were successfully developed and validated. In 24 Murray cod individuals, the number of alleles per locus ranged from 9 to 14 (mean=11.58), while the effective number of alleles ranged from 5.111 to 11.805 (mean=8.415). The observed heterozygosity ranged from 0.273 to 0.833 (mean=0.554), whereas the expected heterozygosity ranged from 0.804 to 0.915 (mean=0.877). The polymorphic information content values ranged from 0.782 to 0.909 (mean=0.864). These results demonstrate that the SSR markers developed in this study exhibit high levels of polymorphism and provide effective molecular tools for population genetic analysis, resource conservation, and marker-assisted breeding of Murray cod.