Twenty polymorphic microsatellite loci were developed via 454 sequencing for two hybridizing sister species of butterflyfish: the spot-band butterflyfish (Chaetodon punctatofasciatus) and peppered butterflyfish (Chaetodon guttatissimus), which are widely distributed in the Western Pacific and Indian Ocean, respectively. All loci were genotyped in samples collected from Christmas Island: C. guttatissimus (n = 25), C. punctatofasciatus (n = 17) and hybrids (n = 16). Mean alleles per locus (N a) were: 9.05 for C. guttatissimus, 9.95 for C. punctatofasciatus and 9.45 for hybrids. Observed heterozygosity (H O) ranged from 0.00 to 1.00 for C. guttatissimus; from 0.08 to 0.88 for C. punctatofasciatus; and from 0.19 to 0.94 for hybrids. Most loci conformed to Hardy-Weinberg expectations, were in linkage equilibrium, and did not contain null alleles. These markers will be useful for testing population genetic hypotheses including patterns of hybridization in this pair of butterflyfishes.