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Fluorescence in situ hybridization (FISH) using oligonucleotide probes for (GAA) 6 (18 bp) and ribosomal DNA (rDNA) (5S rDNA, 41 bp) was applied to analyse Zanthoxylum armatum. (GAA) 6 loci were detected on the pericentromeric regions of five chromosome pairs, and 5S rDNA loci were also detected on the pericentromeric regions of another two chromosome pairs. The densities and locations of (GAA) 6 and 5S rDNA signals varied between individual chromosomes. High-intensity (GAA) 6 signals were detected at the centromeres of two large and two smaller metacentric chromosomes. Relatively strong (GAA) 6 signals were detected at the centromeres of two relatively small metacentric chromosomes, although strong 5S rDNA signals were detected at the centromeres of two additional smaller metacentric chromosomes. Weak (GAA) 6 signals were detected at the centromeres of four large metacentric chromosomes, whereas weak 5S rDNA signals were detected at the centromeres of two smaller metacentric chromosomes. The remaining chromosomes exhibited no signals. Zanthoxylum armatum had 2n = ∼128. The lengths of the mitotic metaphase chromosomes ranged from 1.22 to 2.34 μm. Our results provide information that may be beneficial for future cytogenetic studies and could contribute to the physical assembly of the Zanthoxylum genome.