Abstract Backgrounds In children with sepsis, circulatory shock and multi-organ failure remain major contributors to mortality. Prolonged capillary refill time (PCRT) is a clinical tool associated with disease severity and tissue hypoperfusion. Microcirculation assessment with videomicroscopy represents a promising candidate for assessing and improving hemodynamic management strategies in children with sepsis. Particularly when there is loss of coherence between the macro and microcirculation (hemodynamic incoherence). We sought to evaluate the association between PCRT and microcirculation changes in sepsis. Methods This was a prospective cohort study in children hospitalized with sepsis. Microcirculation was measured using sublingual video microscopy (capillary density and flow and perfused boundary region [PBR]—a parameter inversely proportional to vascular endothelial glycocalyx thickness), phalangeal tissue perfusion, and endothelial activation and glycocalyx injury biomarkers. The primary outcome was the association between PCRT and microcirculation changes. Results A total of 132 children with sepsis were included, with a median age of two years (IQR 0.6–12.2). PCRT was associated with increased glycocalyx degradation (PBR 2.21 vs. 2.08 microns; aOR 2.65, 95% CI 1.09–6.34; p = 0.02) and fewer 4–6 micron capillaries recruited (p = 0.03), with no changes in the percentage of capillary blood volume (p = 0.13). Patients with hemodynamic incoherence had more PBR abnormalities (78.4% vs. 60.8%; aOR 2.58, 95% CI 1.06–6.29; p = 0.03) and the persistence of these abnormalities after six hours was associated with higher mortality (16.5% vs. 6.1%; p