In this study, multicolor FISH analysis on metaphase chromosomes of spinach with biotin-labeled 25S rDNA, DIG-labeled telomere sequences and biotin-labeled and DIG-labeled 5S rDNA was performed. There were six 25S rDNA loci, which were located on the satellites of the third, the fifth and the sixth chromosomes, four 5S rDNA loci, which were located on the long arms of the third and the fifth chromosomes. The telomere loci were located on the end of the sixth chromosome and also on both the end and centromeric regions of other chromosomes. This study is an important complement to both traditional karyotype analysis and FISH karyotype analysis in spinach.