Pentapedal locomotion is the use of the tail as a fifth leg during the slow gait of kangaroos. Although previous studies have informally noted that some smaller species of macropodines do not engage in pentapedal locomotion, a systematic comparative analysis of tail use during slow gait across a wide range of species in this group has not been done. Analysis of relative movement of the pelvis, tail, and joint angles of the lower limbs during slow gait, using 2D landmark techniques on video recordings, was carried out on 16 species of Macropodinae. We also compared the relative lengthening of the tibia using crural index (CI) to test whether hindlimb morphology was associated with pentapedal locomotion. Pentapedal locomotion was characterised by three features: the presence of the ‘tail repositioning phase’, the constant height of the pelvis and the stationary placement of the distal tail on the ground during the hindlimb swing phase. The mean CI of pentapedal species was significantly greater than that of non-pentapedal species (1.71 versus 1.36; P < 0.001). This lends support to the hypothesis that the use of pentapedal locomotion is associated with the relative lengthening of the hindlimb, which in turn is associated with body size and habitat preference within the Macropodinae.