The African baobab (Adansonia digitata L.), also referred to as the "Tree of Life", is a majestic, long-lived and multipurpose tree of sub-Saharan Africa. Internationally, a growing demand for baobab products in the food, pharmaceutical and cosmetics industries has been observed. Considering this, there is a need for scientific information on the genetics and breeding of A. digitata, including cytogenetics, genetic diversity and reproductive biology. The objectives of our cytogenetic research were to determine the genome size, chromosome number, and organization of ribosomal DNA (45S and 5SrDNA) of A. digitata. Flow cytometry analysis revealed a 2C-DNA value of 3.8 ± 0.6 pg (1Cx monoploid genome size 919.1 ± 62.9 Mbp). Using our improved chromosome preparation technique, we were able to unequivocally count the chromosomes resulting in 2n = 4x = 168, a revised chromosome number for A. digitata. Fluorescent in situ hybridization (FISH) analysis revealed two massively large variants of 45S rDNA and their corresponding nucleolus organizer regions (NOR). The NOR variants were about two to four times larger than the main body of their respective chromosomes. To our knowledge, this is the first report of this phenomenon in a plant species. Furthermore, we found that FISH analysis using the Arabidopsis-type telomere repeat sequence probe clarified and confirmed the new chromosome number and characterized the 45S rDNA structural organization.