Background: Mesothelin (MSLN) is highly expressed in several malignancies including colorectal cancer (CRC) compared to normal tissue and is associated with tumor cell proliferation and worse patient survival. MSLN could be a potential target for antigen-specific therapy. Here, we characterized the MSLN expression in CRC and its association with molecular and immune-related markers. Methods: 15,673 CRC samples were tested by NGS (592, NextSeq; WES, NovaSeq), WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). Microsatellite-instability (MSI) was tested by fragment analysis, IHC, and NGS. Tumor mutational burden (TMB) totaled somatic mutations per tumor (high>10 mt/MB). Tumors with MSLN-high(H) and MSLN-low(L) expression were classified by top and bottom quartile, respectively. Real world overall survival (OS) was extracted from insurance claims and calculated using Kaplan-Meier estimates for molecularly defined cohorts from tissue collection to last contact. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q<0.05). Results: There was no difference in median age or gender distribution between MSLN-H and MSLN-L CRC tumors. Right-sided tumors had increased median MSLN expression compared to rectal, transverse, and left-sided tumors (6.12 vs 5.29 vs 5.35 vs 4.81 TPM, q<0.05). Median MSLN expression was higher in metastatic compared to primary tumors (1.21-fold, q<0.05). Median MSLN expression was highest in CMS4 (10.17 TPM), but, in MSS CRC, MSLN expression was highest in CMS1 (10.46 TPM, all q<0.05). MSLN-H tumors had higher frequency of KRAS (59.8% vs 36.4%) and BRAF (13.1% vs 9.2%) mutations but lower frequency of APC mutations (67.5% vs 77.2%), TMB High (9.8% vs 12%) and dMMR/MSI-H (6.6% vs 8.9%) compared to MSLN-L tumors (q<0.05). Further, MSLN-H tumors had higher MAPK activation score (MPAS) compared to MSLN-L tumors (1.72-fold, q<0.05). Similarly, in the MSS only cohort, MSLN-H tumors had increased frequency of KRAS (62.2% vs 37.9%)BRAF (10.5% vs 5.0%) mutations but decreased APC mutations (70% vs 80.9%) (q<0.05). MSLN-H tumors had higher expression of immune checkpoint genes (CD274, PDCD1, PDCD1LG2, CTLA4, LAG3, HAVC2 and FOXP3, FC: 1.1-1.8) and T cell inflamed score (3.3-fold) (all q<0.05). MSLN-H was associated with worse OS compared to MSLN-L in all CRC (934 vs 1253 days; HR: 1.21, 95% CI (1.10-1.32), p<0.0001) and MSS CRC (972 vs 1168 days; HR: 1.15, 95% CI (1.06-1.23), p<0.001) but not in MSI-H CRC (p=0.67). Conclusions: Our data suggest a strong association between MSLN expression and increased mutations in the RAS pathway and MAPK activation score, decreased mutations in WNT pathway genes, a differentially modulated immune tumor microenvironment, and poor overall survival. These findings support MSLN as a potential prognostic biomarker candidate in colorectal cancers pending further prospective validation.