Somatic chromosomes of a wild relative of sugarcane (Saccharum spontaneum L.) anther culture-derived clone (AP 85-361, 2n=32) were identified and characterized by computer-aided imaging technology and molecular cytological methods. The presence of four satellite chromosomes and four nearly identical chromosome sets suggests that the clone is a tetrahaploid with the basic number x=8. A quantitative chromosome map, or idiogram, was developed using image analysis of the condensation pattern (CP) at the prometaphase stage of somatic chromosomes. The 45S and 5S ribosomal RNA gene (rDNA) loci were simultaneously visualized by multi-color fluorescence in situ hybridization (McFISH) and precisely localized to the regions of 3p3.1 and 6q1.3 on the idiogram. The simultaneous visualization of two sets of four ribosomal RNA genes confirms tetraploidy of this clone. This conclusion is consistent with results of molecular marker mapping. The quantitative chromosome map produced will become the foundation for genome analyses based on chromosome identity and structure. Previously impossible identification of small chromosomes and untestable hypotheses about the polyploid nature of plants can now be settled with these two approaches of quantitative karyotyping and FISH.