Background: Differentially-methylated regions (DMRs) are characteristic of colorectal cancer (CRC) and some occur more frequently than common mutations. This study aimed to evaluate the clinical utility of assaying circulating cell-free DNA for methylation in BCAT1, IKZF1 and IRF4 for detection of CRC. Methods: A multiplexed real-time PCR assay targeting DMRs in each of the three genes was developed. Assay accuracy was explored in plasma specimens banked from observational cross-sectional trials or from volunteers scheduled for colonoscopy or prior to CRC surgery. Results: 1620 specimens were suitable for study inclusion including 184 and 616 cases with CRC and adenomas, respectively, and 820 cases without neoplasia (overall median age, 63.0 years; 56% males). Combining the PCR signals for all targeted DMRs returned the best sensitivity for CRC (136/184, 73.9%, 95% CI 67.1–79.7), advanced adenomas (53/337, 15.7%, 95% CI 12.0–20.1) and high-grade dysplastic (HGD) adenomas (9/35, 25.7%, 95% CI 14.0–42.3) with a 90.1%, specificity for neoplasia (739/820, 95% CI 87.9–92.0, p < 0.01). Detection of methylation in all three genes were more likely in CRC cases than those without it (OR 28.5, 95% CI 7.3–121.2, p < 0.0001). Of the 81 positive cases without neoplasia, 62 (76.5%) were positive by a single PCR replicate only and predominantly due to detection of methylated BCAT1 (53.2%). Single replicate positivity was significantly higher than that in CRC (26/136, 19.1%, p < 0.0001), and single BCAT1 replicate positivity was more likely in cases without neoplasia than in CRC (OR 17.7, 95% CI 6.6–43.3, p < 0.0001). When a positive result was limited to those with ≥ 1 PCR replicate positive for either IKZF1 or IRF4, or at least two replicates positive for BCAT1, the multi-panel test maintained a high sensitivity for CRC (131/184, 71.2%, 95% CI 64.3–77.3) and HGD adenomas (8/35, 22.9%, 95% CI 11.8–39.3, p = 0.029) but improved specificity significantly (772/820, 94.1%, 95% CI 92.3–95.6, p < 0.0001 vs. any PCR replicate positive). Conclusion: The multi-panel methylation assay differentiates cases with CRC from those without it and does so with high specificity when criteria for BCAT1 detection are applied. The marker panel is flexible and studies in those at average risk for CRC are now warranted to determine which panel configuration best suits screening goals. Trial registration: ACTRN12611000318987. Registered 25 March 2011, https://www.anzctr.org.au/ ACTRN12611000318987.