Co-occurring and mutually exclusive gene alteration events are helpful for understanding carcinogenesis but systematic screening for such events is quite limited. We conducted pairwise-screening tests to identify “hit pairs” in colorectal cancer (CRC) by utilizing the cross-omics data from the Cancer Genome Atlas (TCGA). Numerous hit pairs involving somatic mutations, CNVs and DNA methylation were found to occur non-randomly in CRC, such as KRAS and HOXB6, SMAD4 and PMEPA1. Based on these hit pairs, we identified 32 synthetic lethal pairs and 7,527 co-occurring pairs relating to drug response. Our further biological experiments showed that the co-occurrence of mutant FCGBP and NUDT12 silencing (or mutant TMC3 and RPS6KA6 silencing) with siRNA reduced cell viability. Moreover, novel hit pairs could influence the prognosis. The patients who carried concurrent mutations of IRF5 and NEFH, SYNE1 and TTN, or MUC16 and NEFH had worse survival outcomes. Particularly, the presence of mutant SYNE1 and TTN pair not only affects prognosis, but also is related to CRC patients’ response to drug treatment. Our “hit-pair” genes may provide insights into colorectal carcinogenesis and help open new avenues for CRC therapy. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.