Nonradioactive amplified fragment-length polymorphism (AFLP) marker detection, a PCR-based, DNA-fingerprinting technique, was achieved by blotting AFLP products after electrophoresis onto a nylon membrane and subsequently hybridizing the blot with an alkaline phosphatase-labeled AFLP probe. Similar AFLP profiles were obtained by both a nonradioactive, chemiluminescent detection technique and by conventional AFLP marker detection using 32P-labeled AFLP primers. The suitability of the method using different gel systems combined with subsequent chemiluminescent detection of AFLP markers is validated by similar dendrograms that were generated using the unweighted pair group method with arithmetic averages (UPGMA). Moreover, chemiluminescent detection of AFLP markers using a universal AFLP nonradioactive probe has been successfully applied on prokaryotes such as Agrobacterium and eukaryotic genomes such as soybean and fungi.