We report the first investigation of nuclear genetic variability in the colony of the spotted seal (Phoca largha) in the Liaodong Gulf, China, where the estimated population size was under 1000 individuals during the study period (2005–2008). A total of 29 microsatellite loci from five pinniped species were employed in 176 spotted seals. Only 15 were polymorphic, with a maximum of 4 alleles detected in samples, with the mean number of alleles per locus being 2.73. Estimates of expected heterozygosity H_e for the polymorphic loci ranged from 0.24 (± 0.05 SD) to 0.72 (± 0.02 SD), with a mean H_e per polymorphic locus of 0.51 (± 0.05 SD). No deviation from the Hardy-Weinberg equilibrium was detected for any of the loci. No differences were detected for either genic or genotypic frequencies of seals from the different sampling sites and years. Several statistical methods were applied to detect the population reduction and compare it with that in other seal species. The result indicated that the population of the Liaodong Gulf spotted seal has suffered a decrease in genetic variability and population reduction over the last several decades. The present study supports a notion that a decrease in population size is the main factor accounting for the low levels of variability observed. These research results provide useful data for the conservation and management of the Liaodong Gulf colony.