Background: Gene fusions involving R-spondin (RSPO) family members have been shown to drive Wnt-dependent tumor initiation in colorectal cancer (CRC). Therapies targeting Wnt pathway are being actively investigated for tumors harboring RSPO2/3 fusions. Here we set out to characterize the molecular features of CRC with and without RSPO fusions to gain insight into potential rationale combination therapy strategies. Methods: Tumor DNA sequencing of 592 genes (NextSeq, Illumina), RNA sequencing of 53 gene fusions (ArcherDx FusionPlex) and immunohistochemistry for PD-L1 on tumor cells (SP142) were tested on CRC tumors at Caris Life Sciences, Phoenix, AZ. Molecular profiles of RSPO2/3 positive (pos) were compared with negative (neg) tumors, Fisher-Exact was used for comparative analysis. Results: A total of 1356 CRC samples were analyzed. RSPO3 and RSPO2 fusions were detected in 42 (3.1%) and 4 (0.3%) samples, respectively, including 5 fusion events not previously reported (e.g., IFNGR1-RSPO3). A female predominance was seen in RSPO fusion pos vs. neg tumors (71.7% vs 45.0%, p < 0.001); no association with age or tumor sidedness was seen. RSPO2/3 fusions were mutually exclusive of MSI-high (0 vs. 5%), ERBB2 alterations (0 vs. 1% mutation, 4% amplification) and other Wnt pathway activation drivers including APC (2 vs. 75%), CTNNB1 (0 vs. 1.4%) and RNF43 (0 vs. 5.3%) mutations. Significantly higher BRAF (26 vs. 7%), RAF1 (4.5 vs. 0.4%) and SMAD4 (30 vs. 11%) mutation rates were seen in RSPO pos vs. neg tumors (p < 0.05). A universal co-activation of MAPK pathway (KRAS, NRAS or BRAF) was seen with RSPO fusions. There was a significantly elevated PD-L1 expression in RSPO3 pos tumors (14%) compared to RSPO neg (6%, p = 0.04) and APC-mutated (5%, p = 0.02) tumors that are MSS. Conclusions: This is the largest series of CRC cases harboring an RSPO rearrangement reported to date. Comprehensive molecular analyses asserted the unique molecular landscape associated with RSPO fusions in CRC and suggested potential combinatorial approach to target Wnt/MAPK pathway. The immune modulatory effects specific to RSPO2/3 fusion revealed by PD-L1 expression suggest co-targeting Wnt pathway with PD1/PDL1 inhibitors in RSPO pos tumors.