Kenaf (Hibiscus cannabinus), a native warm-seasonal crop in Africa, is being considered for genetic improvement for local bast fiber production. To expedite its genetic improvement through breeding, kenaf genotypes from Ghana were assessed for genomic diversity regarding their chromosomal composition and ploidy levels. To gain insight into the repetitive DNA fractions in kenaf, the organization of 5S and 35S rRNA genes, as well as telomeric signal patterns were studied by a molecular cytogenetic approach. Using multi-color fluorescent in situ hybridization, distinct rDNA loci and Arabidopsis-like telomere signal patterns were revealed. The 5S rRNA genes were conserved in kenaf and localized in interstitial regions of two chromosomes across all accessions. The 35S rRNA genes were variable across the kenaf accessions and localized at sub-terminal ends and rarely interstitially in eight or six chromosome arms. Telomeric signals were observed at terminal ends of all chromosomes and the chromosome configuration of Ghana kenaf accessions were each confirmed to be 2n = 2x = 36. Observed genomic instability in kenaf is discussed. This report provides cytogenetic insights into the genome organization of kenaf and variability of its breeding lines. Additionally, this study sets the basis for further research to analyze the repetitive DNA sequences and develop reference karyotypes to reveal genetic and evolutionary relationships between cultivated and wild Hibiscus species.