Understanding population connectivity and genetic diversity is of fundamental importance to conservation. However, in globally threatened marine megafauna, challenges remain due to their elusive nature and wide-ranging distributions. As overexploitation continues to threaten biodiversity across the globe, such knowledge gaps compromise both the suitability and effectiveness of management actions. Here, we use a comparative framework to investigate genetic differentiation and diversity of manta rays, one of the most iconic yet vulnerable groups of elasmobranchs on the planet. Despite their recent divergence, we show how oceanic manta rays (Mobula birostris) display significantly higher heterozygosity than reef manta rays (Mobula alfredi) and that M. birostris populations display higher connectivity worldwide. Through inferring modes of colonization, we reveal how both contemporary and historical forces have likely influenced these patterns, with important implications for population management. Our findings highlight the potential for fisheries to disrupt population dynamics at both local and global scales and therefore have direct relevance for international conservation of marine species.