In recent years, the incidence of colorectal cancer (CRC) under the age of 50 years has increased significantly and population-based screening is currently not offered to persons under 50 years. Consequently, persons with early-onset (<50 years) CRC (EOCRC) more frequently present with symptomatic disease at advanced stages (III/IV) resulting in greater loss of life in young cases. In 2020, the U.S. Preventive Services Task Force recommended the age for CRC screening by colonoscopy or fecal tests be reduced to 45 years. However, the uptake of screening by these methods in all screen-eligible populations is low, including in those <50 years who are genetically at high risk, so adherence in the asymptomatic population <50 years is also likely to be low. Also, over half of EOCRC occur in persons under <45 years of age, where no such screening would be offered. A minimally invasive, blood-based screening test for EOCRC would provide a cost-effective and patient-friendly option for triaging and identifying those warranting a follow-up colonoscopy, while increasing screening adherence in the younger population. Cancer-specific methylated DNA biomarkers are highly suited for population-based cancer screening via the detection of circulating tumor DNA (ctDNA) in blood plasma because they are more prevalent across patients with a given cancer type than tumor mutations and are more stable (nuclease-resistant) in plasma. The methylated SEPTIN9 plasma ctDNA test, “mSEPT9” (Epi proColon V2.0, Epigenomics), is FDA-approved for CRC screening in persons aged ≥50 years who decline colonoscopy and fecal tests. This test urgently needs to be assessed in persons <50 years to determine its suitability for the detection of EOCRC. Even so, a multi-marker test is likely to have superior sensitivity to a single-marker test. Thus, the objectives of our study are to identify and confirm a panel of methylated ctDNA markers for the plasma-based detection of EOCRC, and to compare the diagnostic performance of this panel to the mSEPT9 test in persons <50 years of age. In Aim 1, we will identify the most prevalent differentially-methylated (tumor vs. normal) DNA markers in CRC cases <50 years by performing deep Methyl-Seq across 4.2 million CpG sites in paired primary tumor vs. normal colon tissues from EOCRC cases, and leukocytes from healthy controls (to filter out non-specific markers). The identified markers will be validated in independent sample series. For the top-ranked markers, we will then develop methylation-specific real-time PCR assays with high analytical sensitivity and test these in pooled plasma from metastatic CRC cases (to select markers producing the strongest signals) and healthy controls (to eliminate any producing low-level non-specific signals) to finalize the marker panel. In Aim 2, we will evaluate the diagnostic performance characteristics of the mSEPT9 test vs. the multi-marker panel test in plasma from colonoscopy-verified CRC cases and controls <50 years. This study will yield a multi-marker methylated ctDNA panel with improved diagnostic performance over the single-marker mSEPT9 test for cost-effective, blood-based, CRC screening in asymptomatic persons <50 years of age.