The onchobothriid tetraphyllidean cestode genus Acanthobothrium van Beneden, 1849, parasitic in the spiral intestine of elasmobranch fishes, was investigated in the Australian region. Thirty-three species are recognised, including 27 that are new. Diagnoses compare the morphological taxonomic characters of all congeners. New species are: Acanthobothrium adlardi; A. angelae; A. arlenae; A. bartonae; A. blairi; A. brayi; A. cannoni; A. chisholmae; A. clarkeae; A. cribbi; A. edmondsi; A. gasseri; A. gibsoni; A. gloveri; A. jonesi; A. lasti; A. laurenbrownae; A. martini; A. mooreae; A. ocallaghani; A. odonoghuei; A. pichelinae; A. robertsoni; A. rohdei; A. stevensi; A. thomasae; and A. walkeri. Additional morphological data are provided for A. australe Robinson, 1965, A. pearsoni Williams, 1962, A. heterodonti Drummond, 1937 and A. urolophi Schmidt, 1973, reported previously from Australia. Acanthobothrium rhynchobatidis Subhapradha, 1955 and A. semnovesiculum Verma, 1928 are reported from Australia for the first time and are redescribed. Additional morphological details are provided for A. ijimae Yoshida, 1917 and A. grandiceps Yamaguti, 1952. Acanthobothrium wedli Robinson, 1959 is redescribed from the type host from New Zealand waters and considered a sister species of A. blairi from Tasmania. Seven new host genera for Acanthobothrium are reported: Hypnos Duméril, 1852 (Hypnidae); Pristiophorus MÜller & Henle, 1837 (Pristiophoridae); Sutorectus Whitley, 1939 (Orectolobidae); Aptychotrema Norman, 1926 and Trygonorrhina MÜller & Henle, 1838 (Rhinobatidae); Parascyllium Gill, 1862 (Parascylliidae); and Aetomylaeus Garman, 1908 (Myliobatididae). Species of Acanthobothrium are reported from the families Hypnidae, Pristiophoridae and Parascylliidae for the first time. New host species for Acanthobothrium are: Pristiophorus cirratus (Latham, 1794); Parascyllium ferrugineum McCulloch, 1911; Sutorectus tentaculatus (Peters, 1865); Aptychotrema vincentiana (Haacke, 1885); Trygonorrhina fasciata MÜller & Henle, 1841; Raja whitleyi Iredale, 1938; Raja cerva Whitley, 1939; Hypnos monopterygium (Shaw & Nodder, 1795); Dasyatis annotata Last, 1987; Urolophus cruciatus (Lacépède, 1804); Urolophus expansus McCulloch, 1916; Urolophus lobatus McKay, 1966; Urolophus paucimaculatus Dixon, 1969; Gymnura australis (Ramsay & Ogilby, 1886); Aetomylaeus nicofii (Schneider, 1801); and Myliobatis australis Macleay, 1881 (Myliobatididae). New host records for Australia include the above 16 elasmobranch species and the following three host species also known to harbour Acanthobothrium in other geographic localities: Rhynchobatis djiddensis (Forsskål, 1775) (Rhynchobatidae); Himantura uarnak (Forsskål, 1775); and Pastinachus sephen (Forsskål, 1775) (Dasyatidae). Four additional records for hosts previously reported for Acanthobothrium from Australian waters are Squalus megalops (Macleay, 1881) (Squalidae), Heterodontus portusjacksoni (Meyer, 1793) (Heterodontidae), Orectolobus maculatus (Bonnaterre, 1788) (Orectolobidae) and Trygonoptera ‘testacea’ MÜller & Henle, 1841 (Urolophidae). An emended diagnosis of the genus, key to Australian species, host-parasite checklist, phylogenetic analysis of the Australian species and an updated world list of all species of Acanthobothrium are provided.