Penetration and non-penetration lap laser welding is the joining method for assembling side facade panels of railway passenger cars, while their fatigue performances and the difference between them are not completely understood. In this study, the fatigue resistance and failure behavior of penetration 1.5+0.8-P and non-penetration 0.8+1.5-N laser welded lap joints prepared with 0.8 mm and 1.5 mm cold-rolled 301L plates were investigated. The weld beads showed a solidification microstructure of primary ferrite with good thermal cracking resistance, and their hardness was lower than that of the plates. The 1.5+0.8-P joint exhibited better fatigue resistance to low stress amplitudes, whereas the 0.8+1.5-N joint showed greater resistance to high stress amplitudes. The failure modes of 0.8+1.5-N and 1.5+0.8-P joints were 1.5 mm and 0.8 mm lower lap plate fracture, respectively, and the primary cracks were initiated at welding fusion lines on the lap surface. There were long plastic ribs on the penetration plate fracture, but not on the non-penetration plate fracture. The fatigue resistance stresses in the crack initiation area of the penetration and non-penetration plates calculated based on the mean fatigue limits are 408 MPa and 326 MPa, respectively, which can be used as reference stress for the fatigue design of the laser welded structures. The main reason for the difference in fatigue performance between the two laser welded joints was that the asymmetrical heating in the non-penetration plate thickness resulted in higher residual stress near the welding fusion line.