According to the transcriptome database of Sepiella japonica, the microsatellites have been explored at a high-throughput scale using a MIcroSAtellite identification tool. In total, 127575 unigenes were obtained after assembly and removal of redundant sequences. The total length of unigenes after sequence splicing was 103104058 bp. The microsatellites in the unigenes were analyzed, and a total of 62124 simple sequence repeats (SSR) (complete SSR, 36813) was identified in 50626 unigenes, accounting for 48.70% of the total unigenes, with one SSR per 16.6 kb. In addition, the microsatellite sequences mainly composed of dinucleotide repeats (31227, 50.27%), followed by trinucleotide repeats (16230, 26.13%). Furthermore, 122 types of repeat motifs were classified in all SSR motifs, (AT/AT)n was the most frequent repeat motif (23.33%), followed by (AAAG/CTTT)n (17.42%). The average length of microsatellites (complete SSR) was 25.87 bp, while that of the microsatellites over 20 bp was 51%. There was a significant negative correlation (P<0.01) between the frequency of microsatellites and the length, the correlation coefficient was ‒0.594. The results of the present study lay a foundation for the development of highly polymorphic microsatellite primers to identify parentage relationship and population genetic diversity, and to evaluate the effect of enhancement and release of S. japonica.