Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the technique of multicolor fluorescence in situ hybridization (M-FISH) for identifying chromosome aberrations in esophageal carcinoma cell line KYSE 410-4, four pools of 6-color whole-chromosome painting probes have been designed and hybridized on the same metaphase spread by four rounds of repetitive FISH. Repetitive 6-color M-FISH was successfully established and the cytogenetic abnormalities in KYSE 410-4 cells were characterized. Chromosome gains occurred at 2q, 3, 8, 17p, and X. An isochromosome 3q was visualized in the cell line, which might be one intermedi- ate mechanism leading to 3p losses and/or 3q gains. Furthermore, 16 structural arrangements were detected, including four derivative chromosomes. The rearrangement of the centromeric regions accounted for approximately 44% of all rearrangements. The results added a more complete and accurate information of the genetic alterations to the classical cytogenetic description of KYSE 410-4 and provided a detailed cytogenetic background data for appropriate use of the cell line. The established 6-color M-FISH was useful for analyzing chro- mosomes in the whole genome of human tumors. Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the technique of multicolor fluorescence in situ hybridization (M-FISH) for identifying chromosome aberrations in esophageal carcinoma cells line KYSE 410-4, four pools of 6-color whole-chromosome painting probes have been designed and hybridized on the same metaphase spread by four rounds of repetitive FISH. Repetitive 6-color M-FISH was successfully established and the cytogenetic abnormalities in KYSE 410-4 cells were characterized. Chromosome gains occurred at 2q, 3, 8, 17p, and X. An isochromosome 3q was visualized in the cell line, which might be one intermedia- ate mechanism leading to 3p losses and / or 3q gains. Furthermore, 16 structural arrangements were detected, including four derivative chromosomes. The rearrangement of the centromeric regions accounted for approxim ately 44% of all rearrangements. The results added a more complete and accurate information of the genetic alterations to the classical cytogenetic description of KYSE 410-4 and provided a detailed cytogenetic background data for appropriate use of the cell line. The established 6-color M-FISH was useful for analyzing chro-mosomes in the whole genome of human tumors.