Background: LNs mets are thought to occur before distant mets. However, lymphatic and distant mets arise from independent subclones of the primary tumor, suggesting that LNs are not essential intermediaries for distant mets. We aimed to comprehensively characterize the molecular profile of LN mets and to explore the differences between LN vs distant mets and primary tumors. Methods: Tumor samples from primary CRCs, LNs, and distant mets were analyzed using NGS (MiSeq on 47 genes, NextSeq on 592 genes), 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. Results: In total, 11871 tumors samples were examined, comprising primaries (N = 5862), distant (N = 5605) and LNs mets (N = 404). The most frequently mutated genes in LNs were TP53 (72%), APC (61%), KRAS (39%), ARID1A (20%), and PIK3CA (12%). LNs showed a higher mean TMB (13 mut/MB) vs distant mets (9, P < .0001). TMB-high (17mut/MB) was more frequent in primaries and LNs vs distant mets (9.5% and 8.8% vs 4.2%, P < .001 and P = .001, respectively), as well as MSI-H (8.8% and 6.9% vs 3.7%, P < .001 and P = .017, respectively). TMB-high is significantly higher in LNs vs distant mets and primaries (P < .0001), independent of MSI-H status. Analyzing distant mets by location, LNs showed higher TMB compared to lung, liver and peritoneum mets (P < .0001). Overall, LNs showed significantly different rates of mutations in APC, KRAS, PI3KCA, KDM6A, and BRIP1 (P < .01 for all comparisons) vs primaries; while presenting a distinct molecular profile compared to distant mets (TP53 72 vs 67%; KRAS 39 vs 50%; RNF43 7 vs 4%; ATM 5 vs 3%; KDM6A 4 vs 1%; BRCA2 4 vs 2%; MSH6 3 vs 2%; PTCH1 4 vs 1%; BRCA1 2 vs 1%; GNAS 2 vs 5%; P < .05 for all comparisons). Our cohort of 30 paired samples confirmed the molecular heterogeneity between primaries, LNs, and distant mets. Conclusions: This is the largest study to investigate the molecular differences between LNs mets, distant mets and primary tumors in CRC patients. Our data support the hypothesis that lymphatic and distants mets harbor different mutation profiles which suggests that they may arise from distinct subclones.