Microsatellite Instability (MSI) status is an established predictive biomarker for the treatment of the anti-programmed death 1 (PD-1) antibody. The current approach to determine the MSI status in tumours requires matched normal DNA. Some mononucleotide microsatellite markers are known to have few variant alleles in both Caucasians and Asians. Therefore, the length of these microsatellite makers is almost confined within the quasi-monomorphic variation range (QMVR). Considering the application of MSI testing for various types of cancers, a simple, sensitive and inexpensive method is desired. This study assessed the clinical utility of the QMVR for determining the MSI status in patients with unresectable metastatic colorectal cancer (mCRC). The study enrolled 435 patients with mCRC. The concordance of the MSI status in mCRC between the standard method using tumour DNA plus matched normal DNA and the testing method using only tumour DNA was evaluated. Eleven (2.5%) MSI-high cases were detected by both the standard and testing methods. The sensitivity and specificity of the testing method were both 100%, indicating complete concordance between the methods. Among the mononucleotide markers, three and two patients showed discordance for NR-21 and BAT-25, respectively. Results from MSI testing with normal tissue indicated that four of five patients had rare germline variants outside the QMVR. For BAT-26, NR-24 and MONO-27, all patients showed complete concordance. Using the QMVR, the MSI status of mCRC can be determined without matched normal DNA.