A non-radioactive 2-deoxyglucose (2DG) analog has been developed here for hyperpolarized magnetic resonance investigations. The analog, [13C6,D8]2DG, showed 13% polarization in solution (27,000-fold signal enhancement at the C1 site), following a dissolution-DNP hyperpolarization process. The phosphorylation of this analog by yeast hexokinase (yHK) was monitored in real-time with a temporal resolution of 1 s. We show that yHK selectively utilizes the β anomer of the 2DG analog, thus revealing a surprising anomeric specificity of this reaction. Such anomeric selectivity was not observed for the reaction of yHK or bacterial glucokinase with a hyperpolarized glucose analog. yHK is highly similar to the human HK-2, which is overexpressed in malignancy. Thus, the current finding may shed a new light on a fundamental enzyme activity which is utilized in the most widespread molecular imaging technology for cancer detection - positron-emission tomography with 18F-2DG.