BackgroundReef manta rays (Mobula alfredi) are globally distributed in tropical and subtropical seas. Their life history traits (slow growth, late maturity, low reproductive output) make them vulnerable to perturbations and therefore require informed management strategies. Previous studies have reported wide-spread genetic connectivity along continental shelves suggesting high gene flow along continuous habitats spanning hundreds of kilometers. However, in the Hawaiian Islands, tagging and photo-identification evidence suggest island populations are isolated despite proximity, a hypothesis that has not yet been evaluated with genetic data.ResultsThis island-resident hypothesis was tested by analyzing whole mitogenome haplotypes and 2048 nuclear single nucleotide polymorphisms (SNPs) betweenM. alfredi(n = 38) on Hawaiʻi Island and Maui Nui (the 4-island complex of Maui, Molokaʻi, Lānaʻi and Kahoʻolawe). Strong divergence in the mitogenome (ΦST= 0.488) relative to nuclear genome-wide SNPs (neutralFST= 0.003; outlierFST= 0.186), and clustering of mitochondrial haplotypes among islands provides robust evidence that female reef manta rays are strongly philopatric and do not migrate between these two island groups. Combined with restricted male-mediated migration, equivalent to a single male moving between islands every 2.2 generations (~ 64 years), we provide evidence these populations are significantly demographically isolated. Estimates of contemporary effective population size (Ne) are 104 (95% CI: 99–110) in Hawaiʻi Island and 129 (95% CI: 122–136) in Maui Nui.ConclusionsConcordant with evidence from photo identification and tagging studies, these genetic results indicate reef manta rays in Hawaiʻi have small, genetically-isolated resident island populations. We hypothesize that due to the Island Mass Effect, large islands provide sufficient resources to support resident populations, thereby making crossing deep channels separating island groups unnecessary. Small effective population size, low genetic diversity, and k-selected life history traits make these isolated populations vulnerable to region-specific anthropogenic threats, which include entanglement, boat strikes, and habitat degradation. The long-term persistence of reef manta rays in the Hawaiian Islands will require island-specific management strategies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12862-023-02130-0.