The golden cuttlefish, Sepia esculenta Hoyle, 1885 (Cephalopoda: Sepiidae) is a valuable and important fishery resource for China, Japan and Korea. This fishery has experienced severe population decline largely due to overexploitation in past decades. To provide guidelines for fisheries management, we estimated genetic diversity and population structure across four locations along the coast of Japan and one location in China (a total 281 individuals) using nine microsatellite DNA loci. Sepia esculenta showed high genetic variability with mean allele richness ranging from 15.8 to 21.4, and mean heterozygosity from 0.80 to 0.90. Weak but significant genetic differentiation was present and the FST value was 0.020 across all five populations. The useful information obtained in this study will offer insights into how to fine-tune conservation and fishery management measures for this species and resource in the future.