This study evaluated whether periodontal ligament (PL) thickness varied with root size and examined the possible influence of this variation on orthodontic mechanics. Measurements were taken of the maxillary left first molar in 54 male Wistar rats. Mean mesial and distal PL thicknesses were compared between the intermediate buccal and mesiobuccal roots using paired Student's t-tests with a 5% significance level. Mean values differed significantly between roots (p < 2.2 × 10-16). PL thickness in rats is directly proportional to root dimensions.