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We sought to assess in-stent variations in fractional flow reserve (FFR) in patients with previous percutaneous coronary intervention (PCI) and to associate any drop in FFR with findings by optical coherence tomography (OCT) imaging. Suboptimal post-PCI FFR values were previously associated with poor outcomes. It is not known to which extent in-stent pressure loss contributes to reduced FFR. In this single-arm observational study, 26 patients who previously underwent PCI with drug-eluting stent or scaffold implantation were enrolled. Motorized FFR pullback during continuous intravenous adenosine infusion and OCT assessments was performed. Post-PCI FFR 0.03 was associated with suboptimal FFR. In patients with suboptimal FFR, the OCT analyses revealed higher mean neointimal area (respectively: 1.06 ± 0.80 vs. 0.51 ± 0.23 mm2; p = 0.018) and higher neointimal thickness of covered struts (respectively 0.11 ± 0.07 vs. 0.06 ± 0.01 mm; p = 0.021). Suboptimal FFR values following stent-implantation are mainly caused by significant in-stent pressure loss during hyperemia. This finding is associated to a larger neointimal proliferation.