The hybrid giant tiger grouper is a fish that has considerable commercial value and has become increasingly important for aquaculture in South East Asia since 2008. In order to prevent any reduction in genetic diversity in hybrid grouper as a result of aquaculture, we have identified 21 microsatellite markers that can be used to estimate genetic variation in the fish population. The number of alleles at polymorphic microsatellite loci ranged from 2 to 7, and observed and expected heterozygosities varied from 0.0323 to 0.9643 and 0.0921 to 0.7174, respectively. Polymorphism information content values ranged from 0.088 to 0.737. Nineteen of the 21 loci were in Hardy-Weinberg equilibrium (P > 0.005) after application of the Bonferroni correction (k = 10), the exceptions being ZZLD35 (P < 0.005) and ZZLD36 (P < 0.001). No linkage disequilibrium was detected. These 21 microsatellite markers are potentially of great value for analyzing genetic diversity to provide essential information for sustainable management of these fish.