Light and scanning electron microscopy have been utilized to further resolve the taxonomic status of the genus Acanthopleura Guilding, 1829 (Mollusca: Polyplacophora) following Ferreira’s 1986 controversial revision, which synonymised four well-established genera and numerous species. Specimens of the 19 nominal species of the genus Acanthopleura, together with those from five widely disparate, geographic populations of one of these species (A. gemmata), along with specimens of the outgroup, Onithochiton quercinus Gould, 1846), have been utilised. A consideration of gross morphological characters, including features of the valves, girdle armature and gills, clearly separate A. rehderi from Acanthopleura, aligning it with Onithochiton. They also suggest the synonymy of two pairs of species (A. haddoni/A. vaillantii and A. testudo/A. brevispinosa), and indicate that A. loochooana is closely aligned with species previously assigned to Squamopleura (A. araucariana, A. curtisiana and A. miles). Examination of microstructural characters of the intermediate valve, including features of the tegmental micro architecture, the ocelli, the aesthetes and the central anterior eaves, confirm the conclusions of the gross morphological study, and, in addition, indicate a close relationship between A. echinata and A. nigra. Investigation of the girdle armature indicates that Acanthopleura can be divided into four groups based on the possession of predominantly scales, spines, spinelets or spicules. However, there is wide intraspecific variation with regard to micro architecture of the girdle elements. This section confirms the close relationships of A. haddoni/A. vaillantii and A. testudo/A. brevispinosa, and suggests affiliations between other species.