Glucuronoxylan is the main hemicellulose in the secondary cell wall of angiosperms. Elucidating its molecular structure provides a basis for more accurate plant cell wall models and the utilization of xylan in biorefinery processes. Here, we investigated the spacing of acetyl, glucuronopyranosyl and galactopyranosyl substitutions on Eucalyptus glucuronoxylan using sequential extraction combined with enzymatic hydrolysis and mass spectrometry. We found that the acetyl groups are preferentially spaced with an even pattern and that consecutive acetylation is present as a minor motif. Distinct odd and even patterns of glucuronidation with tight and sparse spacing were observed. Furthermore, the occurrence of consecutive glucuronidation is reported, which adds to the growing body of evidence that this motif is not only present in gymnosperms but also in angiosperms. In addition, the presence of terminal galactopyranosyl units, which can be released by β-galactosidase, altered the digestibility of the glucuronoxylan by GH30 and GH10 xylanase and appeared to be clustered within the polymeric backbone. These findings increase our understanding of the complex structure of glucuronoxylans and its effect on the extractability and biological degradation of Eucalyptus wood.