High rates of mutation and homoplasy mean that microsatellites generally are not considered to be useful molecular markers for inferring systematic relationships between species. However, an earlier pilot study suggested that conserved flanking microsatellite sequences, also known as repetitive flanking sequences (ReFS), may form a basis for a dominant marker that can differentiate between species of Lepidoptera. We present data that demonstrate that ReFS are quick and easy to use, and generate highly repeatable banding patterns from a range of Lepidoptera species. Sequence data from a subset of ReFS-amplified bands revealed microsatellite families with flanking sequences that are more conserved within than among species: this is probably attributable to recombination-mediated events, transposition of mobile elements or a combination of the two. Our data support the use of ReFS as dominant interspecific molecular markers, and add to the growing literature on the evolution of microsatellites in Lepidoptera.