The tiger shark (Galeocerdo cuvier) occupies tropical and warm-temperate coastal and shelf waters throughout the world, and like many large elasmobranch species, faces various anthropogenic threats, particularly fishing-related mortality.The population structure and genetic diversity of G. cuvier is unknown and data allowing evaluation of the effect of current levels of exploitation of the species is lacking. Mitochondrial DNA sequence data obtained from two molecular markers (control region and NADH dehydrogenase subunit 2) was used to investigate the genetic population structure of the tiger shark across its range in Australia as well past and present genetic diversity. Historical DNA extracted from old tiger shark jaws enabled analysis of temporal changes in genetic diversity. Contemporary tiger shark samples were obtained from within the Coral and Tasman Seas on the east coast, the Indian Ocean on the west coast and the Timor and Arafura Seas in the north. Concatenated sequence data from 86 contemporary G. cuvier samples revealed 12 haplotypes to use for population structure analysis. Analysis of molecular variance identified significant population structure between East and West (ΦST = 0.07879, P <0.05) and between West and North (ΦST = 0.07989, P <0.05). Genetic diversity at the control region was higher in the West than the East in both modern and historical samples; however, a temporal decline in diversity on the east (Historic; h=0.262, p=0.00403 and Modern; h=0.152, p=0.00027) was identified using invaluable information from historical DNA. Overall genetic diversity was low compared to other shark species.This study is among the first to provide crucial baseline data concerning the genetic status of tiger sharks in Australian waters. The presented findings may have significant implications for fisheries management and conservation of G. cuvier given the evidence of low and declining genetic diversity in eastern Australian waters.