This study aimed to define the variability in the microstructure of Norway spruce within an annual ring by examining differences between earlywood and latewood. In particular, we were interested in obtaining new information on bordered pit occurrence and locations relative to tracheid ends, plus the lumina dimensions and longitudinal overlap of tracheids that collectively define the longitudinal hydraulic pathways. A stacked series of X-ray micro-CT scans of an annual ring of Norway spruce were made and stitched together longitudinally to form a three-dimensional volume. The imaging resolution was carefully chosen to capture both longitudinal and transverse anatomical details. Measurements of tracheid length, overlap, radial lumen diameter, and bordered pit location were made semi-automatically using image analysis. The distribution of radial lumen diameter was used to define earlywood and latewood. Then bordered pit linear density and spatial distribution, tracheid length and overlap were analysed, presented and contrasted for earlywood and latewood. Further differences between earlywood and latewood were found only in bordered pit linear density. Clear trends in radial lumen diameter and pit linear density were observed with radial position within the growth ring. These results provide new information on the variability of the Norway spruce microstructure within an annual ring.