The quest for a universal and efficient method of identifying species has been a longstanding challenge in biology. Here, we show that accurate identification of species in all domains of life can be accomplished by multiplex analysis of variable-length sequences containing multiple insertion/deletion variants. The new method, called SPInDel, is able to discriminate 93.3% of eukaryotic species from 18 taxonomic groups. We also demonstrate that the identification of prokaryotic and viral species with numeric profiles of fragment lengths is generally straightforward. A computational platform is presented to facilitate the planning of projects and includes a large data set with nearly 1800 numeric profiles for species in all domains of life (1556 for eukaryotes, 105 for prokaryotes and 130 for viruses). Finally, a SPInDel profiling kit for discrimination of 10 mammalian species was successfully validated on highly processed food products with species mixtures and proved to be easily adaptable to multiple screening procedures routinely used in molecular biology laboratories. These results suggest that SPInDel is a reliable and cost-effective method for broad-spectrum species identification that is appropriate for use in suboptimal samples and is amenable to different high-throughput genotyping platforms without the need for DNA sequencing.