The bitter leaf plant (Vernonia amygdalina Delile) is an indigenous shrub tree species highly cultivated in West and Central Africa for its nutritional and medicinal values. We used 10 random amplified polymorphic DNA (RAPD) markers to assay 30 accessions of V. amygdalina Delile ecotypes (bitter and less bitter leaves, respectively) collected from the southern states of Nigeria (Cross River, Edo, and Oyo). The results obtained showed that RAPD markers were highly polymorphic (98.0%) and generated a total of 29 PCR bands -ranging from two for OPH-03 to 10 for OPB-01 primers. The polymorphic information content was highest for the OPB-01 primer (0.768). Moreover, gene diversity (H e = 0.800) was high, and cluster analysis delineated the accessions into seven groups, which indicated that a significant genetic diversity was present among the accessions studied. The geographical distinctness observed among the accessions indicated a possible effect of plant isolation by distance and a restricted gene flow. The results obtained in this study showed a genetic variability that could be exploited for varietal delineation and used to improve this indigenous species in Nigeria. This is the first report on the molecular study of genetic diversity in V. amygdalina.