Yellowfin tuna are a high value pantropically distributed tuna species managed as a single stock within the Indian Ocean. While studies to date have not provided evidence that a revision to this single stock assumption is warranted, further exploring and understanding the level of population heterogeneity is a priority for sustainable management of these fisheries. This paper presents results from a recent investigation of population structure of yellowfin tuna using cutting-edge sequencing technology as part of a larger collaborative project “Population Structure of IOTC species and sharks of interest in the Indian Ocean (PSTBS-IO)”. A total of 1206 individuals from 9 Indian Ocean areas and two outlier locations (east Atlantic Ocean and south-west Pacific Ocean) were collected. The samples consist of a mix of YoY fish and mature adults, with predominantly YoY in the equatorial regions and adult fish in the sub-tropical and temperate regions. A total of 664 samples, matched to the intended sampling design of the study, were chosen to be sequenced using DArTSeq and included in the analysis of population structure and examination of population connectivity. Model selection criteria using StockR indicate that 2 genetic groupings within the Indian Ocean are more likely than 1, with the likelihood for 1 and 3 groups being similar. Samples collected for this project, which are representative of major fishing areas within the Indian Ocean, were composed of a minimum of two (but likely more) genetically differentiated groups of yellowfin tuna. The most prominent difference is evident between groups sampled north and south of the equator. The fish sampled north of the equator may consist of at least two genetic groups. The samples from the southern regions cannot be statistically differentiated into more than a single group, although there is some indication of more than one group in these regions. In addition, population analysis of the two outgroups of yellowfin tuna show evidence of restricted gene flow, indicating the Indian Ocean is genetically isolated from the Atlantic and Pacific Oceans, which are likely the result of environmentally induced physiological barriers to migration.