Background High on-treatment platelet reactivity (HTPR) has been shown to be associated with adverse cardiac events in patients undergoing percutaneous coronary intervention, but the effect of baseline hematologic parameters upon platelet reactivity remains controversial. Objective The present study aims to evaluate the impact of hematocrit on 2 different assay methods used to assess on-treatment platelet reactivity to clopidogrel. Methods We tested clopidogrel on-treatment platelet reactivity in 466 consecutive patients using VerifyNow P2Y12 (VN) and light transmission aggregometry (LTA) with adenosine diphosphate (ADP) 5 and 20 μM assays 6 to 24 hours after percutaneous coronary intervention. Patients were categorized into 4 groups according to baseline hematocrit. One-year major adverse cardiac events, including death, nonfatal myocardial infarction, and definite stent thrombosis, were collected. Results Lower hematocrit was associated with higher P2Y12 reaction unit (PRU) and a higher rate of HTPR (P < .001) as measured by VN assay. No differences were seen among the 4 groups in platelet reactivity measured by LTA using ADP 5 μM (P = .23) and ADP 20 μM (P = .21). In a multivariable logistic regression model, baseline hematocrit was independently associated with PRU ≥208 (odds ratio [OR] 0.92, 95% CI 0.86-0.97, P = .005) but had no correlation with LTA ADP 5 μM ≥46% (OR 1.0, 95% CI 0.95-1.06, P = .88) or LTA ADP 20 μM ≥59% (OR 1.03, 95% CI 0.97-1.09, P = .39). In a logistic regression model, the addition of VN assay results, hematocrit, and interaction between the hematocrit and assay results had shown a significant influence on the area under curve for prediction of 1-year major adverse cardiac events compared with baseline clinical variables only for PRU (0.63 vs 0.76, P = .006) but not with LTA (0.64 vs 0.74, P = .13). Conclusion Baseline hematocrit has a differential influence on results of the ex vivo platelet functional assays. Lower baseline hematocrit was independently associated with HTPR by VN but not LTA. This may affect the interpretation of platelet function testing in patients with significant anemia.