Poly(ethylene 2,5-furandicarboxylate) (PEF) is an emerging bio-based polymer with interesting thermal and barrier properties. In this study, the melting behavior of PEF was investigated in situ by means of simultaneous wide and small angle X-ray scattering (WAXS and SAXS) measurements coupled with DSC measurements. This study gives the first evidence of what happens from a structural point of view during the multiple melting behavior of PEF, which is composed of three distinct events, taking into account the nature of the initial crystalline phase present. The first result is that the alpha ' form, induced at low crystallization temperature, does not undergo any phase transformation upon heating revealing its stable character. Second, the comparison of the SAXS and WAXS results with the DSC ones showed that the multiple melting behavior observed is attributed to a melting-recrystallization-melting process. Third, this work also definitely shows that the low amplitude melting endotherm observed in the DSC thermograms is ascribed to the melting of secondary crystals. Finally, SAXS-WAXS results led to the conclusion that the secondary crystals cannot be depicted by the commonly accepted lamellar insertion model. Another microstructural representation of these secondary crystals is proposed. In this model, the secondary crystals consist of bundles of macromolecules, which formed small crystalline entities located between the primary crystalline lamellae stacks.