Abstract Background Anti-EGFR antibody is one of the options for treatment of RAS wild-type metastatic colorectal cancer (mCRC), especially when the primary tumor site locates in the left sided large intestine. We have shown that methylation status evaluated by genome-wide DNA methylation test correlates with clinical outcomes of anti-EGFR treatment in RAS wild-type mCRC. Here, we report about the development of novel in vitro diagnostics (IVD) based on DNA methylation status of selected CpG sites. Method We retrospectively collected KRAS wild-type mCRC cases who received anti-EGFR treatment in salvage line. DNA methylation status was measured for 16 CpG sites by our novel IVD using real-time PCR method for each case. Based on DNA methylation status, they were classified into 2 groups. Highly-methylated colorectal cancer (HMCC) was defined when 8 or more sites among 16 sites were methylated and low-methylated colorectal cancer (LMCC) was classified when it was less than 8 sites. We compared the clinical outcomes between the 2 groups, and the predictive accuracy of the DNA methylation status by the new diagnostic method was examined. Results A total of 83 cases were collected, with 20 cases classified as HMCC and 63 cases classified as LMCC. The response rate of the anti-EGFR treatment was 5.0% (1/20 cases) in HMCC and 30.2% (19/63 cases) in LMCC, respectively (p = 0.032). The median progression-free survival were 2.8 months and 5.3 months, respectively (p Conclusion DNA methylation status of the 16 CpG sites measured by our novel IVD was significantly correlated with the therapeutic effect of anti-EGFR treatment for KRAS wild-type mCRC. Currently, extended analysis is in progress using the data of more than 200 cases. We will report the results including the analysis of RAS status and primary tumor site.