AbstractLymph nodes (LNs) and distant metastases can arise from independent subclones of the primary tumor. Herein, we characterized the molecular landscape and the differences between LNs, distant metastases and primary colorectal cancers (CRCs). Samples were analyzed using next generation sequencing (NGS, MiSeq on 47 genes, NextSeq on 592 genes) and immunohistochemistry. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated by NGS of known MSI loci. In total, 11,871 samples were examined, comprising primaries (N = 5862), distant (N = 5605) and LNs metastases (N = 404). The most frequently mutated genes in LNs were TP53 (72%), APC (61%), KRAS (39%), ARID1A (20%), PIK3CA (12%). LNs showed a higher mean TMB (13 mut/MB) vs distant metastases (9 mut/MB, p < 0.0001). TMB-high (≥17mut/MB) and MSI-H (8.8% and 6.9% vs 3.7%, p < 0.001 and p = 0.017, respectively) classifications were more frequent in primaries and LNs vs distant metastases (9.5% and 8.8% vs 4.2%, p < 0.001 and p = 0.001, respectively). TMB-high is significantly more common in LNs vs distant metastases and primaries (P < 0.0001), regardless MSI-H status. Overall, LNs showed significantly different rates of mutations in APC, KRAS, PI3KCA, KDM6A, and BRIP1 (p < 0.01) vs primaries, while presenting a distinct molecular profile compared to distant metastases. Our cohort of 30 paired samples confirmed the molecular heterogeneity between primaries, LNs, and distant metastases. Our data support the hypothesis that lymphatic and distant metastases harbor different mutational landscape. Our findings are hypothesis generating and need to be examined in prospective studies.