Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome-derived SSRs (CG-SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty-six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG-SSRs showed that these populations did not deviate from linkage equilibrium or Hardy–Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG-SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.