Background:Identification of high-risk stage II colorectal cancer (CRC) patients, potential candidates for adjuvant chemotherapy, is challenging. Current clinical guidelines rely mainly on histopathological markers with relatively weak prognostic value. This motivates further search for prognostic markers.Methods:This explorative study aimed to identify potential candidate gene mutations to facilitate differentiation between subgroups of patients with CRC stage II. Panel-based massive parallel sequencing was used to genetically characterize tumor tissues from 85 patients radically operated for CRC stage II, of which 12 developed recurrent cancer during follow-up. Genetic data was compared between patients with or without cancer recurrence, between tumors located in colon and in rectum, and for association with tumor differentiation grade.Results:Genetic variation inATM,C11ORF65was associated with recurrence-free survival. Previous reports regarding the association betweenBRAFmutation and a higher age at diagnosis, and tumor location in colon were confirmed.APC,BRAF, orKRASmutation was associated with tumor differentiation grade. Multiple correspondence analyses revealed no obvious clustering of patients with the studied clinical characteristics, indicating that the genetic signatures observed here were unique for each individual.Conclusions:Taken together, we have demonstrated the utility of panel-based massive parallel sequencing to explore the pathogenesis of CRC stage II. We have identified promising candidate gene mutations associated with cancer recurrence, tumor location, and differentiation grade in patients with CRC stage II, which merit further investigation.