Bladder cancer is a common neoplasm worldwide, consisting mainly of transitional cell carcinomas, while squamous, adenocarcinoma, and sarcomatoid bladder cancers account for the remaining cases. In the present study, multiplex fluorescence in situ hybridization (M-FISH) has been used to characterize chromosome rearrangements in eight transitional and one squamous cell carcinoma cell line, RT112, of UMUC-3, 5637, CAT(wil), FGEN, EJ28, J82, 253J, and SCaBER. Alterations of chromosome 9 are the most frequent cytogenetic and molecular findings in transitional cell carcinomas of all grades and stages, while changes of chromosomes 3, 4, 8, 9, 11, 14, and 17 are also frequently observed. In the present study, alterations previously described, including del(8)(p10), del(9)(p10), del(17)(p10), and overrepresentation of chromosome 20, as well as several novel findings, were observed. These novel findings were a del(15)(q15) and isochromosome 14q, both occurring in three of nine cell lines examined. These abnormalities may reflect changes in bladder tumor biology. M-FISH represents an effective preliminary screening tool for the characterization of complex tumor karyotypes.