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The β subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which exists as two isoforms (β1 and β2) in humans, has a carbohydrate-binding module (CBM) that interacts with glycogen. Although the β1- and β2-CBMs are structurally similar, with strictly conserved ligand-contact residues, they show different carbohydrate affinities. β2-CBM shows the strongest affinity for both branched and unbranched oligosaccharides and it has recently been shown that a Thr insertion into β2-CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why β2-CBM binds all carbohydrates with stronger affinity. Herein, it is shown that residue 134 (Val for β2 and Thr for β1), which does not come into contact with a carbohydrate, appears to account for the affinity difference. Characterisation by NMR spectroscopy, however, suggests that mutant β2-Thr101Δ/Val134Thr differs from that of β1-CBM, and mutant β1-Thr101ins/Thr134Val differs from that of β2-CBM. Furthermore, these mutants are less stable to chemical denaturation, relative to that of wild-type β-CBMs, which confounds the affinity analyses. To support the importance of Thr101 and Val134, the ancestral CBM has been constructed. This CBM retains Thr101 and Val134, which suggests that the extant β1-CBM has a modest loss of function in carbohydrate binding. Because the ancestor bound carbohydrate with equal affinity to that of β2-CBM, it is concluded that residue 134 plays an indirect role in carbohydrate binding.