Fractional flow reserve (FFR) measurement was compared to dobutamine stress echocardiography (DSE) instable angina (SA) with stable coronary lesion (s) (SCL (s) ) in a few trials; however, similar comparisons in patients with acute coronary syndrome (ACS) with non-culprit lesion (s) (NCL (s) ) are lacking. Our objectives were to prospectively evaluate the diagnostic performance of FFR with two different cutoff values (< 0.80 and < 0.75) relative to DSE in moderate (30%-70% diameter stenosis) NCLs (ACS group) and to compare these observations with those measured in SCLs (SA group). One hundred seventy-five consecutive patients with SA (n = 86) and ACS (n = 89) with 225 coronary lesions (109 SCLs and 116 NCLs) were enrolled. In contrast to the ACS cohort in SA patients, normal DSE was associated with higher FFR values compared to those with abnormal DSE (P = 0.051 versus P = 0.006). In addition, in the SA group, a significant correlation was observed between DSE (regional wall motion score index at peak stress) and FFR (r = -0.290; P = 0.002), whereas a similar association was absent (r = -0.029; P = 0.760) among ACS patients. In the SA group, decreasing the FFR cutoff value (< 0.80 versus < 0.75) improved the concordance of FFR with DSE (70.6% versus 81.7%) without altering its discriminatory power (area under the curve; 0.68 versus 0.63; P = 0.369), whereas in the ACS group, concordance remained similar (69.0% versus 71.6%) and discriminatory power decreased (0.62 versus 0.51; P = 0.049), respectively. In conclusion, lesion-specific FFR assessment may have different relevance in patients with moderate NCLs than in patients with SCLs.