Misalignments and distortions in welded plate components act as stress raisers and can significantly decrease the fatigue strength of welded connections. This paper investigates the effect of the plate misalignment of transverse attachments on the fatigue behavior of axially-loaded non-load-carrying cruciform (NLCX) joints. Experimental fatigue tests with and without the plate misalignment are carried out for fillet-welded NLCX joints. The test specimens were fabricated of S1100 ultra-high-strength steel grade, and the fatigue tests were conducted using an applied stress ratio of R = 0.1. Numerical finite element analyses were conducted to obtain the stress concentrations induced by the misalignment. In addition, varied geometry parameters were applied to investigate their effect on the magnitude of stress concentrations. Stress concentrations were obtained using the structural hot spot stress method applying linear surface extrapolation and 1 mm below depth methods, and the effective notch stress concept with the reference radius of 1 mm. Experimental fatigue tests showed a decrease of up to 12% in fatigue strength depending on the degree of misalignment. The highest stress concentration was induced when the misalignment to the joint width ratio was e/L = 0.2–0.4. Structural stresses cannot be estimated using the linear surface extrapolation. Instead, structural stress at the 1 mm depth and effective notch stress concept accurately evaluated the misalignment effect on the fatigue performance of NLCX joints, and provided a good correspondence between the theoretical and experimental fatigue strength estimations.