This study aimed to systematically reveal the distribution patterns of repeat sequences in genomic microsatellites of Cynoglossus semilaevis and analyze the functional annotations，providing sequence resources for developing microsatellite molecular markers with potential functional implications and exploring genetic breeding.

Based on the whole-genome sequences of Cynoglossus semilaevis，the microsatellite repeat sequences in genome were screened by bioinformatic methods. GO functional annotation and KEGG signaling pathway enrichment analysis were conducted on genes associated with microsatellites in exonic regions.

A total of 473776 microsatellite loci belonging to six base repeat types were identified in the Cynoglossus semilaevis genome，accounting for 1.62% of the total genome length；the number of microsatellites decreased with the increase in the base number of repeat units，and mononucleotide and dinucleotide microsatellites accounted for 84.85% of all microsatellites，with a significant positive correlation between chromosome length and number of contained microsatellites （P<0.05，the same below）. Among different repeat motif types in genomic microsatellites of Cynoglossus semilaevis，the A/T motif was the most abundant in mononucleotide repeats，and the AC/GT motif was the most abundant in dinucleotide repeats，and the numbers of each type of microsatellite decreased with the increase of motif repeat times. The significantly enriched GO functional terms of genes containing microsatellites in genomic exonic regions of Cynoglossus semilaevis were regulation of transcription by RNA polymerase II and protein phosphorylation. The KEGG signaling pathway enrichment analysis results showed that the significantly enriched signaling pathways for genes containing microsatellites in genomic exonic regions of Cynoglossus semilaevis included starch and sucrose metabolism within the metabolism category and necroptosis within the cellular process，followed by the tight junction within the cellular process.

Genomic microsatellites of Cynoglossus semilaevis are mainly composed of mononucleotide and dinucleotide repeats，and a significant positive correlation is found between the number of microsatellites and chromosome length，with the number decreasing as motif repeat number rises. Microsatellites in exonic regions are significantly enriched in signaling pathways associated with transcriptional regulation，energy metabolism，and immune response，and they probably participate in the adaptive regulation of Cynoglossus semilaevis under environmental stresses such as hypoxia.