The last few years have witnessed rapid development of highly ordered and reproducible surface-enhanced Raman scattering (SERS) nanostructured substrates for their potential medical and analytical application such as biosensing and bioimaging. In this work, 5-nm silver films deposited on nanostructured Al and Al2O3 templates are investigated as substrates for SERS. The chosen templates show different honeycomb nanostructures with two sets of dimension, i.e. pore diameter of ca. 25 and 50 nm and interpore distance of ca. 56 and 100 nm. The SERS imaging results reveal that the signal of the probe molecule (4-thiazolidinone-2-thione) is distributed inhomogeneously on the substrate surface, and this fact is correlated with the morphology of nanostructures determined by atomic force microscopy. The variation of SERS intensity among the substrates is strongly correlated with the shape and size of potential SERS-active sites, e.g. nanocups and nanopores. The strongest SERS response is found for the Ag/Al2O3 template anodized in sulfuric acid, which represents the nanopore array with the smallest dimensions (e.g. pore diameter, interpore distance etc.). Furthermore, depending on size and nanostructure shape, changes in the adsorption mechanism of the probe molecule are observed. Copyright © 2014 John Wiley & Sons, Ltd.