Our understanding of the genetic connectivity of manta ray populations and the drivers that shape genetic population structure is still limited. This information is crucial to identify the spatial boundaries of discrete populations and guide decisions on units to conserve. In this study, we used genome-wide single nucleotide polymorphisms (SNPs) to assess the genetic structure and diversity of reef manta rays Mobula alfredi at a local scale within New Caledonia and regionally in the western Pacific Ocean. We provide the first evidence of fine-scale genetic differentiation in M. alfredi , found between the 3 cleaning station aggregation sites in New Caledonia (n = 65 samples, N = 2676 SNPs, F ST = 0.01, p < 0.0001). Furthermore, population structure was evident at the regional scale between individuals from New Caledonia (n = 73) and East Australia (n = 19) on the basis of genetic differentiation statistics (3619 SNPs, F ST = 0.096, p < 0.0001) and clustering algorithms, with unidirectional gene flow detected from east (New Caledonia) to west (Australia). These results reveal that reef manta rays can form genetically distinct groups within a relatively small geographic range and highlights the need to consider genetic structure when designating management units for conservation action and planning.