The South American genus of Cichlid fish Apistogramma comprises over 100 species, most of which are difficult to identify. There is a need to clarify species limits and evolutionary relationships, conduct fine-scale phylogeographic revision of some species complexes, and collect information on population conservation status. In addition, recent studies suggest that female mate choice may lead to reproductive isolation and potentially to sympatric speciation in some species. Highly variable bi-parentally inherited molecular markers are needed to determine reproductive units in order to test this hypothesis. Microsatellite markers were developed for fine-scale population genetic analyses within the genus Apistogramma, based on a DNA library of 7567 candidate loci. A set of 40 loci with dinucleotide repeats was selected and tested for amplification and dimorphism in three Apistogramma species (six samples). Thirteen polymorphic loci, combined in three multiplex sets, were retained and analyzed in nine Apistogramma species (47 samples) representative of the genus diversity in the Peruvian Amazon basin. All loci presented a high level of heterozygosity and polymorphism in most species. This will be helpful in evolutionary genetics and conservation studies.