Multi-scale analyses were carried out on polyamide 66 (PA66) fibres by single fibre mechanical tests, scanning electron microscopy observations, wide-angle X-ray diffraction (WAXD) characterisation coupled with profile fitting procedure and μ-Raman extensometry. The study has attempted to reveal the microstructural parameters controlling tensile mechanical performance (i.e. stiffness and damage), to describe the micro-mechanisms of deformation involved during loading and finally to suggest a microstructural model. The main role played by isotropic amorphous regions during the first steps of deformation and the initiation of enthalpic deformation modes at higher strain levels were observed either by WAXD or μ-Raman spectroscopy. A crystallization process, induced by mechanical loading, was noted. The final deformation regime, described as viscoplastic, was seen to be activated beyond a threshold strain-value and to be due to macrofibril sliding. These coupled analyses have revealed the discontinuous character of the microfibrillar structure and have enabled a microstructural model for the fibre to be proposed.