Background: We initiated a nationwide screening project in Japan to detect rare cancer-related genome alterations in CRC (GI-SCREEN 2013-01-CRC) or non-CRC GI cancer and to facilitate the enrollment of patients (pts) in clinical trials for targeted therapies (Shitara Ket, al ASCO 2015 TPS4134). In this sub-study, we evaluated relationship between MSI status and oncogenic genome alterations as well as clinicopathological features. Methods: Pts with advanced CRC who are or will be treated with systemic chemotherapy were eligible. A total of 36 mutations of KRAS, NRAS, BRAF, PIK3CA in genomic DNA from FFPE tumor specimens were simultaneously analyzed by Luminex (xMAP) technology. Using the MSI Analysis System (Promega) composed of 5 mononucleotide markers, MSI status was analyzed with or without paired DNA from FFPE normal tissue. Results: Among 853 pts enrolled from Feb. 2014 to Jan. 2015, 839 tumor samples have been analyzed for both oncogenic genome alterations and MSI. Mutations in KRAS exon 2, other KRAS or NRAS, BRAF and PIK3CA were detected in 34.0%, 8.0%, 4.6%, and 8.6%, respectively. MSI status was evaluable in 805 pts, of which MSI-H was detected in 15 pts (1.9%). These MSI-H were also detected without normal tissue in this study. Among MSI-H pts, 6 were associated with BRAF V600E mutation, 3 with KRAS exon 2 mutation, and 2 with PIK3CA mutation (overlapping with KRAS exon2). BRAF+MSI-H was associated with older age at presentation and worse OS than non-BRAF+MSI-H (median age, 71.5 vs. 47yrs; median OS, 6.9 vs. 18.1 months).We also found rare germline variants in microsatellite of NR21 and BAT25, and these allele frequencies were 0.33% and 0.25%, respectively. Conclusions: The frequency of MSI-H advanced CRC was 1.9% in Japan. BRAF+MSI-H was associated with worse prognosis. MSI test and multi-oncogenes mutation analysis were feasible without normal tissue, although rare variant of microsatellite markers may affect the decision of MSI status.