Microsatellite instability (MSI) in colorectal cancer (CRC) occurs in 10-15% of tumours in which reduced expression/function of mismatch repair enzymes MLH1, PMS2, MSH2 or MSH6 compromises the replication of mono- or di-nucleotide repeats. However, a distinct subset of up to 50% of the remaining CRCs exhibit elevated microsatellite instability at (selected) tetra-nucleotide repeats (EMAST), which results from aberrant MSH3 function. These CRCs are associated with poor prognosis. Although not recognised using current immunohistochemistry / microsatellite analysis methods, EMAST CRCs would be included in MSI-L (MSI-low) or MSS (microsatellite stable) groups. In this study, we have optimised methods to detect EMAST-positive CRCs in pathology practice and evaluated the methods using formalin-fixed paraffin-embedded (FFPE) archival CRC blocks. Aberrant MSH3 immunohistochemistry presents as either complete or focal loss of MSH3 expression and/or predominantly cytoplasmic (as opposed to nuclear) localisation of MSH3 in tumour cells. Abnormal MSH3 expression was evident in 62% (31/50) CRCs, however it is apparent that specialised development of scoring methods and pathologist training would be required in order to integrate MSH3 immunohistochemistry into routine pathology workflows. Evidence of tetranucleotide MSI was assessed by PCR/capillary electrophoresis for 7 tetranucleotide repeats using DNA extracted from FFPE CRC and adjacent non-malignant colon tissues. Instability at >1 of the tetranucleotide microsatellites was detected in 44% (35/80) cases. Recent reports have identified that the nuclear-to-cytoplasmic displacement of MSH3 and EMAST occur in response to inflammation, a known and modifiable risk factor for CRC development and progression. Thus, identification of EMAST CRC in clinical practice has manifold applications including the subclassification of non-MSI-H CRC to demarcate patients with poor prognosis tumours who may benefit from additional treatment, surveillance or anti-inflammatory medication (e.g. aspirin). Post hoc analysis of clinical trials to identify EMAST CRCs may also distinguish treatments that better target this poor prognosis subtype.