Microsatellite instability (MSI) testing is tricky in gynaecological cancers (GC). Thus, we aimed to describe the instability patterns to improve MSI test interpretation in sporadic and hereditary GCs. Ninety-five cases, including uterine and ovarian cancers, with known genetic and immunohistochemical (IHC) features, were analysed for MSI by a mononucleotide repeats pentaplex (MRP). We identified 13 ambiguous cases that did not fully meet MSI criteria (‘borderline’ cases, B-MSI), which were mainly represented by MSH2/MSH6-deficient and Lynch syndrome cases. Also, we evaluated nine additional loci of candidate MSI markers that did not improve the detection of MSI cases, but might be useful for discordant or borderline samples. In conclusion, although MSI and IHC test are highly concordant, a subset of ambiguous MSI cases deserves a careful interpretation in particular when MSH2/MSH6 are involved. RPL22 and SRPR testing may be useful to integrate MRP panel for the analysis of critical cases.