Amplified fragment length polymorphism (AFLP) is a genotyping technique based on PCR amplification of specific restriction fragments from a particular genome. The methodology has been extensively used in plant biology to solve a variety of scientific questions, including taxonomy, molecular epidemiology, systematics, population genetics, among many others. The AFLP share advantages and disadvantages with other types of molecular markers, being particularly useful in organisms with no previous DNA sequence knowledge. In eukaryotic pathogens, the technique has not been extensively used, although it has the potential to solve many important issues as it allows the simultaneous examination of hundreds or even thousands of polymorphic sites in the genome of the organism. Here we describe the main applications published on the use of AFLP in eukaryotic pathogens, with emphasis in species of the groups fungi, protozoa and helminths, and discuss the role of this methodology in the context of new techniques derived from the advances of the next generation sequencing.