A low dietary cation-anion difference (DCAD) in forages fed to dry dairy cows decreases the risk of hypocalcaemia. We determined the effect of stages of development at harvest and N fertilization on the DCAD of the spring growth of timothy (Phleum pratense L.). Four stages of development (stem elongation, early heading, late heading, and early flowering) and four rates of N fertilization (0, 60, 120, and 180 kg N ha^-1) were evaluated in a factorial split plot design at Lévis, Québec, Canada. Concentrations of K, Na, Ca, Mg, Cl, S, P, and N, and dry matter (DM) yield of timothy were determined. The short [(K⁺ + Na⁺) - (Cl^- + S^2-)] and long [(K⁺ + Na⁺ + 0.15 Ca²⁺ + 0.15 Mg²⁺) - (Cl^- + 0.6 S^2- + 0.5 P^3-)] DCAD were calculated. Between stem elongation and early flowering, the short DCAD decreased from 326 to 196 meq kg^-1 DM whereas the long DCAD went from 297 to 181 meq kg^-1 DM. This is mainly caused by a decrease of 17% in K concentration with stages of development associated with an increase of 14% in Cl concentration of timothy. Nitrogen fertilization significantly increased the short and long DCAD only at stem elongation; the absence of a response at later stages of development is explained by the concomitant increase in both K and Cl concentrations. Harvesting timothy at late heading or later is therefore suggested to decrease its DCAD, but this practice alone is not sufficient to produce a forage with the target short DCAD of -50 meq kg^-1 DM.