Background: Activation of ROS1 and ALK tyrosine kinases through gene fusions lead to unchecked cell proliferation and transformation. ROS1 and ALK gene fusions were found in about 5% and 2% of lung adenocarcinomas and are highly sensitive to specific tyrosine kinase inhibitors. This study aimed at identifying the presence of ROS1 and ALKrearrangements in CRC using FISH technology. Methods: Arrayed specimens of metastatic CRC (mCRC) patients were tested with a 4-target, 4-color break-apart FISH probe set (Abbott Molecular) designed to simultaneously evaluate the genomic status of ROS1 and ALK. Fused 3’/5’ signals of each gene were considered negative for rearrangement; single 3’/single 5’ (for ROS1) and split 3’-5’ or single 3’ (for ALK) were considered positive for rearrangement. Results: Among 236 mCRC patients tested, two were positive for ROS1 (single 3’ROS1 signals in 39% and 61% of tumor cells) and one was positive for ALK rearrangement (single 3’ALK in 41% of tumor cells). The upper cut-off for positive FISH patterns in the negative specimens was identified as <15% both for ROS1 and ALK. Interestingly, the ALK+ patient displayed intra-tumoral heterogeneity, detected in the tissue cores and confirmed in two resection blocks. The fusion partner for ALK was identified as EML4 by PCR-based tests and sequencing. The fusion partner(s) for ROS1 remains to be identified by other technologies. A small fraction of specimens presented duplicated or clustered copies of native ALK and ROS1. Conclusions: The novel FISH probe set was effective to identify the first cases of ROS1 rearrangements in CRC and re-confirm the occurrence of ALK rearrangements. This supports further evaluation of mCRC cases for ROS1 and ALK gene fusions as these may represent new targets for evaluation in clinical trials. Tumor heterogeneity in the ALK rearrangement must be addressed for screening tests. (Partially supported by research grant from Abbott Molecular and the Colorado CCSG P30CA046934).