Bambara groundnut (Vigna subterranea (L.) Verdc), an African indigenous legume, is popular in most parts of Africa. The present study was undertaken to establish genetic relationships among 16 cultivated bambara groundnut landraces using fluorescence-based amplified fragment length polymorphism (AFLP) markers. Seven selective primer combinations generated 504 amplification products, ranging from 50 to 400 bp. Several landrace-specific products were identified that could be effectively used to produce landrace-specific markers for identification purposes. On average, each primer combination generated 72 amplified products that were detectable by an ABI Prism 310 DNA sequencer. The polymorphisms obtained ranged from 68.0 to 98.0%, with an average of 84.0%. The primer pairs M-ACA + P-GCC and M-ACA + P-GGA produced more polymorphic fragments than any other primer pairs and were better at differentiating landraces. The dendrogram generated by the UPGMA (unweighted pair-group method with arithmetic averaging) grouped 16 landraces into 3 clusters, mainly according to their place of collection or geographic origin. DipC1995 and Malawi5 were the most genetically related landraces. AFLP analysis provided sufficient polymorphism to determine the amount of genetic diversity and to establish genetic relationships in bambara groundnut landraces. The results will help in the formulation of marker-assisted breeding in bambara groundnut.