Yellowfin tuna of the Indian Ocean is overfished, and a better understanding of the stock structure is needed to enable sustainable management. Here, otolith δ18O values of young-of-the-year fish from known nursery areas of the equatorial Indian Ocean (West, Central and East) were used to establish a reference isotopic signature to predict the origin of sub-adult and adult individuals. Sub-adult tuna otolith δ18O values from Reunion Island were similar to the West nursery signature, but otolith δ18O values of sub-adults from Pakistan were unlike any of the nurseries sampled. Therefore, δ18O values from the Pakistan area samples were considered an additional nursery source for predicting the origin of adult tuna, using a multinomial logistic regression classification method. The western equatorial area was the most productive nursery for three fishing grounds sampled, with a minor contribution of Pakistan-like origin fish. Contribution of Central or East nurseries to the adult population was negligible. One adult otolith was analysed by secondary ion mass spectrometry along the otolith growth transect and results were compared with an isoscape approach to infer lifetime movements. This study is an important first step towards understanding the spatial structure and connectivity of the species.