Detection of inter-layer and internal defects in semiconductor silicon (Si) wafers by non-contact, non-destructive and depth-resolving techniques with a high lateral and depth resolution is one of the challenging tasks in modern semiconductor industry. In this paper, we report that nonlinear optical harmonic generation can be of great virtue therein because it enables non-invasive inspection of inter-layer defects with sub-micrometer depth resolution in extensive penetration depth over several millimeters. Compared to existing inspection methods for inter-layer defects, such as ultrasound, photoacoustic and photothermal imaging, the proposed technique provides higher lateral and depth resolution as well as higher interfacial selectivity. For in-depth understanding of nonlinear harmonic generation at Si wafer surfaces, the spectral power distributions of third and fifth harmonics from Si wafers with various crystal orientations and dopants were carefully analyzed under different incident polarizations and excitation depths using a near-infrared (NIR) femtosecond laser as the excitation light source. We finally demonstrated that inter-layer defects inside stacked Si wafers, such as delamination or stacking faults, can be inspected with a high lateral and depth resolution in a non-contact and non-destructive manner. These findings will pave the way for nonlinear optical harmonic generation to the fields of interfacial studies of crystalline materials, high-resolution detection of sub-diffraction-limit surface defects, and high-resolution imaging of internal structures in stacked semiconductor devices.