Mitochondrial DNA (mtDNA) polymorphisms provide useful information that can be used to estimate the phylogeographic relationships, historical demography, and migratory events of widely distributed animals. In this study, the spatio-temporal patterns of mtDNA polymorphisms were assessed in Indo-Pacific hawksbill turtles from Malaysian nesting rookeries and foraging aggregations sampled during 1996–2014. Clear genetic differences were observed between turtles from the Sabah Turtle Islands rookery in the Sulu Sea, and the rookeries of Melaka and Redang Island in the South China Sea off the Malay Peninsula; however, no temporal genetic changes were found to have occurred on a decadal time scale. Despite the descriptive evaluation of a few samples, Johor nesting turtles possessed different haplotypes from those at the proximate Melaka rookery, indicating the importance of conserving this small nesting population. Although continuous sampling efforts are needed to determine the relationships between specific rookeries and foraging aggregations, the presence of multiple haplotypes in Malaysian foraging aggregations, in combination with Lagrangian drifter buoy data, suggests that there have been migrations to foraging grounds in Southeast Asia from various rookeries. This study provides basic information for the conservation and management of hawksbill turtles in Southeast Asia.