The most effective protocol for cardiovascular screening of competitive athletes remains highly controversial. This study was a prospective, multicenter trial of cardiovascular screening at 35 National Collegiate Athletic Association institutions. Screening included a standardized history and physical examination (PE) as recommended by the American Heart Association and a 12-lead electrocardiogram (ECG) at rest. Centralized electrocardiographic interpretation was provided using the Seattle criteria. Athletes with screening abnormalities underwent additional evaluation directed by the host institution medical team. Primary outcomes included the proportion of total and false-positive screens; the sensitivity, specificity, and positive predictive value of history, PE, and ECG; and the prevalence of serious cardiovascular disorders associated with severe morbidity or sudden cardiac death. From August 2012 to June 2014, 5,258 athletes from 17 intercollegiate sports were screened: 55% men (mean age 20.1 years), 73% Caucasian, 16% African-American, and 11% other/mixed race. At least 1 positive cardiac symptom or family history response was reported by 1,750 athletes (33.3%). PE was abnormal in 108 athletes (2.1%), and electrocardiographic abnormalities were present in 192 athletes (3.7%). Thirteen athletes (0.25%) were identified with serious cardiac conditions including hypertrophic cardiomyopathy (1), large atrial septal defect with right ventricular dilation (1), and ventricular pre-excitation (11). The false-positive rate for history was 33.3%, PE 2.0%, and ECG 3.4%. The sensitivity/specificity/positive predictive value for history was 15.4%/66.9%/0.1%, PE 7.7%/98.2%/0.9%, and ECG 100%/96.6%/6.8%. In conclusion, electrocardiographic screening in National Collegiate Athletic Association athletes has a low false-positive rate and provides superior accuracy compared with a standardized history and PE to detect athletes with potentially dangerous cardiovascular conditions.