Cola spp., one of the most important tree crops, has great nutritional, medicinal, and industrial value. The implementation of molecular markers has demonstrably expedited breeding programs aimed at cultivating diverse crop varieties. These markers facilitate efficient selection for desirable traits, thereby reducing the time and resources required for traditional breeding methodologies. Consequently, the development and deployment of improved crop cultivars have been substantially accelerated through this technology. Molecular marker techniques are considered to be the most suitable means for estimating genetic diversity. This study undertook a molecular characterization of 40 Kola accessions sourced from germplasm collections. The objective was to assess the extent of genetic diversity present within this sample, providing insights into the germplasm’s genetic structure. Inter simple sequence repeat (ISSR) markers were used to assay 40 accessions of Kola collected from the Cocoa Research Institute of Nigeria (CRIN) Ibadan germplasm. The results demonstrated that the ISSR markers were highly polymorphic and generated alleles ranging from nine to twenty-seven. The polymorphic information content was highest for UBC834, and the mean average was 0.832. The observed high gene diversity (0.946) suggests substantial genetic variation within the studied population. UPGMA dendrogram analysis of ISSR markers corroborated this finding, revealing a similarity coefficient range of 46–100%, further confirming the presence of considerable variability. These results collectively indicate a genetically diverse population structure. The results revealed that ISSR markers are useful for genetic characterization as they provide information on the interspecies phylogenetic status of the accessions and reveal genetic variability that could be exploited for varietal delineation and used to improve this species in Nigeria.