In this article, results will be presented on how different x‐ray mask substrate materials affect the quality of the alignment signal acquired through them. By affecting the signal quality, the different materials ultimately have a significant impact on the capability of the alignment system to discern the position of the alignment marks precisely. Data will also be presented on position measurement repeatability as a function of mark depth for the different mask materials. In the experiments, in‐house grown SiC and diamond films were used. Vendor SiC and SiN materials were also used. For comparison and reference, signals were acquired through polyimide and B‐doped Si as well. The alignment was done with a lab‐based Suss ALX100 system. Since the primary alignment effect of membrane materials is to reduce signal‐to‐noise ratio, mark recognition repeatability and signal contrast was used as the comparative performance criteria. Experiments were done in both bright field and nonscanning dark field modes from alignment marks with depths between 5 and 150 nm. The effects of the substrate material became more evident when it was attempted to align using shallow marks. The vendor SiC and diamond exhibited poor recognition repeatability when marks of less than 45 nm in depth were used in bright field or 75 nm in dark field. Antireflective coatings seem useful in improving the performance of some of these materials, especially in bright field alignment. With a one sigma repeatability budget of 10 nm, current alignment systems may have difficulties in reproducibly locating shallow marks (below 20 nm), which may be found on some of the very demanding levels, when aligning through any of the candidate materials. The need may exist for careful processing of the marks and/or improvement in the aligners ability to see through rough or highly absorbing mask materials.