Abstract Aims Diagnosis of acute myocardial infarction (AMI) can be challenging in patients with prior coronary artery bypass grafting (CABG). Methods and results Final diagnoses were adjudicated by two independent cardiologists using the universal definition of AMI among patients presenting to the emergency department (ED) with suspected AMI. Diagnostic accuracy of 34 chest pain characteristics (CPCs) and four electrocardiogram (ECG) signatures stratified according to the presence or absence of prior CABG were prospectively quantified. Among 4015 patients (no prior CABG: n = 3686; prior CABG: n = 329), prevalence of AMI and unstable angina were higher in patients with prior CABG (35% vs. 18%; 26% vs. 8%; both P < 0.001). Three CPCs (9%) and two electrocardiographic findings (50%) showed a different diagnostic performance (interaction P < 0.05) with loss of diagnostic value in patients with prior CABG. The diagnostic accuracy as quantified by the area under the curve (AUC) of the integrated clinical judgement was moderate to good in patients with prior CABG, and significantly lower compared to patients without prior CABG [AUC 0.80 (95% confidence interval (CI) 0.75–0.84) vs. AUC 0.87 (95% CI 0.86–0.89); P = 0.004]. Time to discharge from the ED was significantly longer in patients with prior CABG [359 (215–525) min vs. 300 (192–435) min; P < 0.001]. Key findings were confirmed in a large independent external validation cohort (n = 13 653). Conclusions Patients with prior CABG presenting with suspected AMI have a high prevalence of AMI and unstable angina and lower diagnostic accuracy of CPCs and the ECG, possibly justifying liberal use of early coronary angiography in these vulnerable patients. ClinicalTrials.gov registry Number NCT00470587.