Fungal copper radical oxidases (CROs) from the Auxiliary Activity family 5 (AA5) constitute a group of metalloenzymes that oxidize a wide panel of natural compounds, such as galactose‐containing saccharides or primary alcohols, into product derivatives exhibiting promising biotechnological interests. Despite a well‐conserved first copper‐coordination sphere and overall fold, some members of the AA5_2 subfamily are incapable of oxidizing galactose and galactosides but conversely efficiently catalyse the oxidation of diverse aliphatic alcohols. The objective of this study was to understand which residues dictate the substrate preferences between alcohol oxidases and galactose oxidases within the AA5_2 subfamily. Based on structural differences and molecular modelling predictions between the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) and the archetypal galactose oxidase from Fusarium graminearum (FgrGalOx), a rational mutagenesis approach was developed to target regions or residues potentially driving the substrate specificity of these enzymes. A set of 21 single and multiple CgrAlcOx variants was produced and characterized leading to the identification of six residues (W39, F138, M173, F174, T246, L302), in the vicinity of the active site, crucial for substrate recognition. Two multiple CgrAlcOx variants, i.e. M4F (W39F, F138W, M173R and T246Q) and M6 (W39F, F138W, M173R, F174Y, T246Q and L302P), exhibited a similar affinity for carbohydrate substrates when compared to FgrGalOx. In conclusion, using a rational site‐directed mutagenesis approach, we identified key residues involved in the substrate selectivity of AA5_2 enzymes towards galactose‐containing saccharides. Fungal copper radical oxidases (CROs) from the AA5_2 subfamily can oxidize a wide panel of natural compounds including galactose‐containing saccharides or primary alcohols, but the molecular determinants driving the substrate specificity of these metalloenzymes were not clearly established. Using a rational site‐directed mutagenesis approach based on molecular modelling predictions, we identified several key amino acids driving affinity towards galactose‐containing substrates.