5-Hydroxymethylfurfural (HMF) is considered an important building block for future bio-based chemicals. Here, we present an experimental study using different ketoses (fructose, sorbose, tagatose) and aldoses (glucose, mannose, galactose) under aqueous acidic conditions (65 g L−1 substrate, 100–160 °C, 33–300 mM H2SO4) to gain insights into reaction pathways for hexose dehydration to HMF. Both reaction rates and HMF selectivities were significantly higher for ketoses than for aldoses, which is in line with literature. Screening and kinetic experiments showed that the reactivity of the different ketoses is a function of the hydroxyl group orientation at the C3 and C4 positions. These results, in combination with DFT calculations, point to a dehydration mechanism involving cyclic intermediates. For aldoses, no influence of the hydroxyl group orientation was observed, indicating a different rate-determining step. The combination of the knowledge from the literature and the findings in this work indicates that aldoses require an isomerization to ketose prior to dehydration to obtain high HMF yields.