Investigation of early urothelial lesions is essential to gain insight into the development of bladder cancer. In order to evaluate if deletions of chromosome arms 9p and 9q are preceding histomorphological changes in the urothelium, histologically normal urothelial samples of patients with previous and/or simultaneous (pre)malignant urothelial lesions were investigated. Dual colour fluorescence in situ hybridization (FISH) was performed on 96 histologically inconspicuous urothelial biopsies from 41 patients. Centromeric probes for chromosomes 9 and 17 were combined with gene locus specific probes for 9p21 (p16/CDKI2), 9q22 (FACC) and 17p (p53). Deletions of chromosome 9 (defined as presence of only one copy of centromere 9 or fewer gene-specific than centromeric signals in at least 40% of evaluated cells) were found in 21% of the samples (18/87). Deletions of chromosome 9p were present in 16% of cases (14/89), whereas deletions of chromosome 9q were encountered in 10% of specimens (9/93). A hemizygous deletion of p53 was found only once. Polysomy in more than 10% of cells was encountered in 16% of cases for both chromosome 9 and chromosome 17. Non-diploidy (defined as polysomy of both chromosomes) was found in 6% of samples (5/80). In summary, chromosome 9 deletions are frequently found in histologically normal urothelium of patients with bladder cancer, although less frequently than in hyperplasias and dysplasias. These findings support the hypothesis of multi-focal development of bladder cancer from histologically inconspicuous but already genetically altered urothelium. FISH analysis of chromosome 9 regions could provide a useful tool to detect potentially premalignant lesions in the follow-up care of patients with bladder cancer.