The MAPK signalling genes KRAS, NRAS and BRAF and the PIK3CA gene are routinely investigated for mutations in the diagnostic routine of colorectal cancer. Few studies have reported co-existing mutations in these genes with clinical relevance, while some have been previously regarded as mutually exclusive. We set to investigate the frequency and co-occurrent mutations in these targets, and the occurrence of mismatch repair deficiency (dMMR) in a large cohort of Danish colorectal cancers. 1000 colorectal tumours were sequenced as part of our diagnostic workflow for KRAS, NRAS, BRAF and PIK3CA mutations using next-generation sequencing (NGS) and analysed by immunohistochemistry (IHC) for loss of the MMR proteins, MLH1, PMS2, MSH2 and MSH6. Co-existing mutations in 12 patients (1.2%) occurred as multiple mutations in the same gene or spread across several genes (KRAS, NRAS and/or BRAF). The frequency of single mutations in the genes occurred with a frequency similar to previously reported, except for a higher frequency of BRAF mutations (18.0%). We found dMMR in 14.6% of the cases with a majority lacking expression of both MLH1 and PMS2. BRAF mutations were only present in dMMR cases involving MLH1 and/or PMS2. Our findings suggest that co-existing mutations occur, except for the hotspot BRAF V600E, which is mutually exclusive with KRAS/NRAS mutations. Therefore, instead of single gene alterations from the MAPK signalling, assessing co-occurrence of mutations within one or more of those genes should also be accounted. This may impact future oncological treatments and should be considered in the diagnostic workflow.