We have recently shown that intrinsic, chromophore free Second Harmonic Generation (SHG) signals can be obtained from myofibrillar structures of mammalian skeletal muscle1,2 (Both et al. 2003, Proc. SPIE 5139: 112-120; Both et al. 2004, JBO 9(5):882-892). Here, we report experiments at the level of single myofibrils (diameters 1 to 2 µm) to characterize the spatial dependency of the hyperpolarizability chi(2) and to generate a map of this tensor in myofibrillar structures. Myofibrils are the smallest functional sub cellular contractile structures of muscle. They are organized in a regular sarcomer pattern with a periodicity of 2 to 3 µm. Single myofibrils were obtained from mammalian skeletal muscle using a combined chemical and mechanical fractionation. The SHG signals were recorded with an inverse laser scanning microscope (Leica SP2). A ps laser source (Ti:Sa laser, Tsunami, Spectra Physics) tuned to 880 nm was used to excite the sample through an objective of high NA (1.2NA, 63x). The laser source was linearly polarized and the axis of polarization could be adjusted in steps of degrees with a half-wave plate. The forward scattered SHG signal was collected with a matching objective placed above the preparation. The SHG signals depend both on polarization and location within the myofibrillar structures. The SHG signals seem to arise from the myosin molecules. In conclusion, SHG imaging allows to monitor the myofibrillar structure with two photon resolution.