The Amplified fragment Length Polymorphism (AFLP) is one of the cost-effective and useful fingerprinting techniques to study non-model species. One crucial AFLP step in the AFLP procedure is the choice of restriction enzymes and selective bases providing good-quality AFLP profiles. Here, we present a user-friendly program (ISIF) that allows carrying out in silico AFLPs on species for which whole genome sequences are available. Carrying out in silico analyses as preliminary tests can help to optimize the experimental work by allowing a rapid screening of candidate restriction enzymes and the combinations of selective bases to be used. Furthermore, using in silico AFLPs is of great interest to limit homoplasy and amplification of repetitive elements to target genomic regions of interest or to optimize complex and costly high-throughput genomic experiments.