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Introduction Lynch syndrome (LS) is an autosomal dominantly inherited form of colorectal cancer (CRC) and is implicated in 2%–4% of CRC cases. LS develops from a mutation in one allele of one of the DNA mismatch repair (MMR) genes, most commonly MLH1 and MSH2, or less frequently MSH6 and PMS2. Loss of functional MMR proteins leads to defects in DNA repair and, subsequently, high DNA microsatellite instability (MSI-High). LS diagnostics currently consists of an analysis of MMR protein expression by means of Immunohistochemistry (IHC) and a molecular analysis to detect MSI. During our 10 year experience in performing LS diagnostics we have encountered few cases where IHC and MSI analyses do not match. This study is to clarify these inconsistent molecular alterations which will contribute to our understanding of LS diagnostics. Material and methods Of 2335 LS cases 27 (1.1%) showed discrepant IHC and MSI results. To clarify this, IHC and MSI analyses were repeated if possible with different antibodies and different MSI markers (mononucleotide instead of dinucleotide markers). Using MS-MLPA MLH1 hypermethylation, BRAF V600E mutation status and LOH of the MMR genes was analysed. Finally, germline and somatic mutations in MMR genes were analysed by using NGS. Protein modelling was also performed to visualise structural protein changes. Results and discussions Among these 27 cases, due to the decline of patients for further testing, 4 cases were excluded from further analyses. Of the remaining cases in 2 cases using a different antibody explained the unexpected results and in 2 cases switching from dinucleotide to mononucleotide MSI markers. In respectively 8 and 4 cases somatic and germline mutations explained the IHC and MSI results. Finally, in 2 cases protein modelling of identified mutations explained the presence of protein staining in MSI-high cases. The rest cases cannot be further studied due to the lack of tissues. Conclusion Based on these results, we have concluded: 1. Mononucleotide markers are more sensitive to detect microsatellite instability. 2. Protein modelling can explain presence of protein staining for mutations that do not cause a major conformational change. 3. Somatic sequencing is a valuable addition to Lynch syndrome diagnostics.