Background: Cardiac troponin I (cTnI) is a more specific and sensitive biomarker than creatine kinase MB (CKMB) for detection of myocardial damage. We report the prevalence of positive cTnI and CKMB mass among patients hospitalized with suspected acute coronary syndrome (ACS) and the potential impact of use of different reference cutoffs, particularly those proposed by European Society of Cardiology/American College of Cardiology (ESC/ACC) consensus guidelines, on rates of diagnosis of acute myocardial infarction (AMI). Methods: We analyzed 1719 consecutive patients with suspected ACS admitted to an urban acute care hospital over a 6-month period. Patients (≥18 years of age) had at least two separate sets of plasma biomarkers (cTnI and CKMB) measured more than 12-24 h after admission to determine the potential rates of AMI based on different biomarker cutoff concentrations. Results: The prevalence of cTnI-positive cases ranged from 10.6%, based on a cutoff of twice the ROC curve (cTnI ≤1.2 μg/L), to 25.0%, using the ESC/ACC-recommended 99th percentile cutoff (cTnI <0.1 μg/L). The prevalence of CKMB-positive cases ranged from 10.4%, with the cutoff of twice the ROC curve (CKMB ≤10.0 μg/L) to 21.7%, with the 99th percentile cutoff (CKMB <3.9 μg/L). Use of the 10% CV cutoff (cTnI ≤0.3 μg/L and CKMB <3.9 μg/L) instead of the ROC cutoff produced a 26% increase in all cTnI-positive cases. Use of the 99th percentile reference cutoff instead of the ROC curve-derived cutoff produced an 85% increase in all cTnI-positive cases. A substantial proportion of the increase in total cTnI-positive cases was derived from cTnI-positive/CKMB-negative cases: 71 (4.1%), 73 (4.2%), 98 (5.7%), and 209 (12.2%) of cTnI-positive cases were CKMB-negative, as determined by the twice the ROC, ROC, 10% CV, and 99th percentile reference cutoffs, respectively. At the 99th percentile cutoffs, 8.8% of cases were CKMB-positive/cTnI-negative. Conclusions: Use of lower reference cutoffs for plasma biomarkers, as recommended by ESC/ACC guidelines, markedly increases the rates of cTnI-positive cases overall. A substantial proportion of the increase in total cTnI-positive cases was derived from the creation of additional cTnI-positive/CKMB-negative cases. CKMB-positive/cTnI-negative cases are likely false positive for myocardial injury.