Alternative splicing enhances proteome diversity and modulates cancer-associated proteins. To identify tissue- and tumor-specific alternative splicing, we have used the GeneChip Human Exon 1.0 ST Array to measure whole-genome exon expression in 102 normal and cancer tissue samples of different stages from colon, urinary bladder, and prostate. We identified 2069 candidate alternative splicing events between normal tissue samples from colon, bladder and prostate and selected 15 splicing events for RT-PCR validation, 10 of which were successfully validated by reverse transcriptase PCR (RT-PCR) and sequencing. Furthermore, 23, 19 and 18 candidate tumor-specific splicing alterations in colon, bladder and prostate, respectively, were selected for RT-PCR validation on an independent set of 81 normal and tumor tissue samples. In total, seven genes with tumor-specific splice variants were identified (ACTN1, CALD1, COL6A3, LRRFIP2, PIK4CB, TPM1 and VCL). The validated tumor-specific splicing alterations were highly consistent, enabling clear separation of normal and cancer samples, and in some cases even of different tumor stages. A subset of the tumor-specific splicing alterations (ACTN1, CALD1 and VCL) was found in all three organs and may represent general cancer-related splicing events. In silico protein predictions suggest that the identified cancer-specific splice variants encode proteins with potentially altered functions, indicating that they may be involved in pathogenesis and hence represent novel therapeutic targets. In conclusion, we identified and validated alternative splicing between normal tissue samples from colon, bladder and prostate in addition to cancer-specific splicing events in colon-, bladder-, and prostate cancer, which may have diagnostic and prognostic implications.