High-throughput DNA sequencing technologies make it possible now to sequence entire genomes relatively easily. Complete genomic information obtained by whole genome resequencing (WGS) can aid in identifying and delineating species even if they are extremely young, cryptic or morphologically difficult to discern and closely related. Yet for taxonomic or conservation biology purposes WGS can remain cost-prohibitive, too time-consuming, and often constitute a “data overkill”. Rapid and reliable identification of species (and populations) that is also cost-effective is possible based on species-specific markers that can be discovered by WGS. Based on WGS data we designed a PCR restriction fragment length polymorphism (PCR-RFLP) assay for 19 Neotropical Midas cichlid populations (Amphilophus cf. citrinellus), that includes all 13 described species of this species complex. Our work illustrates that identification of species and populations (i.e., fish from different lakes) can be greatly improved by designing genetic markers using available “high resolution” genomic information. Yet, our work also shows that even in the best-case scenario, when whole-genome resequencing information is available, unequivocal assignments remain challenging when species or populations diverged very recently, or gene flow persists. In summary, we provide a comprehensive workflow on how to design RFPL markers based on genome re-sequencing data, how to test and evaluate their reliability, and discuss the benefits and pitfalls of our approach.