The genetic similarities of 49 accessions of bananas from The National Banana Collection at Rubona were investigated using random amplified polymorphic DNA markers. A total of 120 primers were screened for their usefulness in amplifying DNA fragments of four cultivars belonging to the subgroup Mutika–Lujugira. Fifteen random primers were selected for more detailed analysis, on the basis of providing reproducible amplification and revealing a high level of variation between the four cultivars. The genetic similarity was estimated using a simple matching coefficient which showed the lowest value of 0.46 between ‘Ingumba’ and ‘Ishika’ and the highest value of 0.85 between ‘Kirayenda’ and ‘Inyabukuwe’. The data of matrix of coefficient of similarity was subjected to cluster analysis using the unweighted pair group method with arithmetic average (UPGMA). Each accession was clearly separated. The results of this study are important for the curation of the banana germplasm collection in Eastern Central Africa and for future breeding of this crop.