The anhydrous crystalline phase of chloroquine bis-(dihydrogen phosphate) is obtained by dehydration of the corresponding hydrate phase, but in common with many solid-state dehydration processes, the product is obtained as a polycrystalline powder, thus limiting the opportunity to carry out structural characterization using single crystal X-ray diffraction techniques. Instead, structure determination of the anhydrous phase has exploited the capabilities of modern powder X-ray diffraction techniques, employing the direct-space Genetic Algorithm technique for structure solution followed by Rietveld refinement. The results reveal that the dehydration process is associated with a significant change in the topology of hydrogen bonded chains of dihydrogen phosphate anions, arising from a change in the hydrogen bonding arrangement within the chains, together with a significant change in the conformation of the chloroquine cation.