AbstractAffinity chromatography is routinely used mostly on a preparative scale to isolate different biomolecules such as proteins and carbohydrates. To this end a variety of proteins is in common use as ligands. To extend the arsenal of binders intended for separation of carbohydrates, we have explored the use of carbohydrate‐binding modules (CBM) in affinity chromatography. The thermostable protein CBM4‐2 and two variants (X‐6 and A‐6) thereof, selected from a newly constructed combinatorial library, were chosen for this study. The CBM4‐2 predominantly binds to xylans but also crossreacts with glucose‐based oligomers. The two CBM‐variants X‐6 and A‐6 had been selected for binding to xylan and Avicel® (a mixture of amorphous and microcrystalline cellulose), respectively. To assess the ability of these proteins to separate carbohydrates, they were immobilized to macroporous microparticulate silica and analyses were conducted at temperatures ranging from 25 to 65°C.With the given set of CBM‐variants, we were able to separate cello‐ and xylo‐oligomers under isocratic conditions. The affinities of the CBMs for their targets were weak (in the mM–µM range) and by adjusting the column temperature we could optimize peak resolution and chromatographic retention times. The access to thermostable CBM‐variants with diverse affinities and selectivities holds promise to be an efficient tool in the field of affinity chromatography for the separation of carbohydrates. Copyright © 2006 John Wiley & Sons, Ltd.