Molecular cytogenetic techniques and computer-aided karyotyping were applied to characterize the chromosomes of spinach (Spinacia oleracea L., 2n = 12). Chromosome lengths, arm ratios, and degrees of condensation at prometaphase chromosomes were analyzed using a software Chromosome Image Analyzing System III (CHIAS III). DNA probes prepared from rice (Oryza sativa L.) rDNA were applied to the spinach chromosomes by the fluorescence in situ hybridization (FISH) method. Three 45S rDNA loci were detected at the nucleolar organizing region (NOR) of Chromosome 5, and at terminal positions of short arms of Chromosomes 2 and 6. The loci of 5S rDNA were also found at three locations. One was at the subtelomeric region of the long arm of Chromosome 2 and the other two were at the proximal region of the long arm of Chromosome 5. All spinach chromosomes were identified which will provide valuable information for mapping genes on these chromosomes.