In this study, CPD (combined PI and DAPI) staining and sequential fluorescence in situ hybridization (FISH) with 45S rDNA probe were applied to analyze the prometaphase and metaphase chromosomes of Caesalpinia decapetala (Roth) Alston, a medicinal plant, and the karyotype of this species was established by combining the chromosome measurements and the CPD bands and rDNA FISH signals. (1) The mean haploid karyotype length was (30.38±1.58) μm. The karyotype formula was 2n=24=14m+10sm (2SAT) with the range of chromosome relative length being 11.22-7.12. The karyotype asymmetry indice, CI, A1, A2, As K(%), TF% and AI were 41.63±6.70, 0.27, 0.16, 58.18, 41.82 and 2.57, respectively, and the asymmetric karyotype belonged to the 2A type of the Stebbins’ category. (2) Four and a half pairs of 45S rDNA sites were identified in the diploid complement. Among these rDNA sites, four pairs were located at the terminals of the short arms of chromosome pairs 3, 8, 9 and 12, respectively; and one site was located at the terminal of the short arm of one homologue of chromosome pair 10, showing heterozygosity of rDNA site. The investigation established the molecular cytogenetic karyotype of C. decapetala for the first time, providing basic data for the genome study of this species.