In grasslands the proportionally largest emission of CO2 comes from the soil. This study aimed to assess how root respiration, a major flux component, is affected by land management and changes in land use. Respiration of roots, separated to classes of different diameter, was measured in 11 temperate mountain grasslands, including meadows, pastures and abandoned sites at three geographic locations. Specific root respiration was affected by nitrogen (N) concentration, root class and land use. The relationship between root N concentration and respiration differed between locations. With increasing root diameter there was a decrease in root respiration, N concentration, respiration per unit N and Q10. In grasslands abandoned for several years specific root respiration was lower than in meadows, pastures and a recently abandoned site. This was due to lower root N concentrations and/or lower respiration rates per unit N within each root class. Since root biomass was higher on abandoned grasslands, total ecosystem root respiration did not differ consistently between sites. Ecosystem root respiration showed distinct seasonal changes due to changes in root biomass, which were less pronounced on abandoned grasslands. Fine roots generally made up the largest portion of ecosystem root respiration, their contribution varying between 35% and 96%. On meadows, clipping increased soil and root respiration by increasing soil temperature. When corrected for temperature effects soil respiration was reduced by 20–50%, whilst root respiration was little affected, suggesting that carbohydrate reserves sustained root metabolism for several days and that microbial respiration strongly responded to short-term changes in assimilate supply.