Satsuma mandarin (Citrus unshiu Marcow.) chromosomes were stained with Giemsa and fluorochromes chromomycin A3 (CMA)/4′,6-diamidino-2-phenyindole (DAPI). Eighteen chromosomes were categorized into eight groups by the position and relative size of the CMA (+) region and relative length of chromosome. Ponkan (C. reticulata Blanco) DNA labeled with Dig-rhodamine (red) and pummelo [C. maxima (Burm.) Merr.] DNA labeled with biotin-fluorescein isothiocyanate (green) were used as genomic in situ hybridization (GISH) probes. GISH signals were detected on CMA (+) regions and other heterochromatin blocks. The chromosomes were categorized into 12 groups by the coloration and size of GISH signals with relative length of chromosomes. GISH allowed six pairs of speculated homozygous and six individual heterozygous chromosomes of satsuma mandarin to be identified unambiguously. In 10 chromosomes with distinct GISH signals on the CMA (+) regions, red GISH signals were detected on nine chromosomes, indicating that satsuma mandarin is closely related to ponkan. Two colors (red and green) of GISH signals were detected on type C chromosome and three different colors (red, green, and yellow) were detected on type A, indicating that pummelo is involved in the origin of satsuma mandarin. The origins of types A and C chromosomes in satsuma mandarin were also discussed. This article demonstrates that GISH is a powerful tool for chromosome identification and karyotyping in citrus.