Arabidopsis hexokinase1 (HXK1) is a moonlighting protein that has separable functions in glucose signaling and in glucose metabolism. In this study, we have characterized expression features and glucose phosphorylation activities of the six HXK gene family members in Arabidopsis thaliana. Three of the genes encode catalytically active proteins, including a stromal-localized HXK3 protein that is expressed mostly in sink organs. We also show that three of the genes encode hexokinase-like (HKL) proteins, which are about 50% identical to AtHXK1, but do not phosphorylate glucose or fructose. Expression studies indicate that both HKL1 and HKL2 transcripts occur in most, if not all, plant tissues and that both proteins are targeted within cells to mitochondria. The HKL1 and HKL2 proteins have 6-10 amino acid insertions/deletions (indels) at the adenosine binding domain. In contrast, HKL3 transcript was detected only in flowers, the protein lacks the noted indels, and the protein has many other amino acid changes that might compromise its ability even to bind glucose or ATP. Activity measurements of HXKs modified by site-directed mutagenesis suggest that the lack of catalytic activities in the HKL proteins might be attributed to any of numerous existing changes. Sliding windows analyses of coding sequences in A. thaliana and A. lyrata ssp. lyrata revealed a differential accumulation of nonsynonymous changes within exon 8 of both HKL1 and HXK3 orthologs. We further discuss the possibility that the non-catalytic HKL proteins have regulatory functions instead of catalytic functions.