Wedgefishes (Rhinidae) are threatened by unsustainable fishing globally, and especially in the Southwest Indian Ocean (SWIO), due to their high-value fins in the shark trade. The whitespotted wedgefish Rhynchobatus djiddensis and the bottlenose wedgefish R. australiae are both classified as Critically Endangered on the IUCN Red List of Threatened Species, yet a lack of species-specific knowledge and taxonomic uncertainty still exists within this genus. Genetic approaches aid in taxonomic classification and identifying distinct populations for targeted conservation. Morphological specimen identification of samples (n = 189) collected across the SWIO was confirmed based on the cytochrome oxidase c subunit I (COI) and/or nicotinamide adenine dehydrogenase subunit 2 (ND2) gene regions. The genetic diversity and population structure within and between species and sampling locations were investigated using a dual marker approach: (1) 2 concatenated mitochondrial gene regions, namely COI and the control region (n = 117), and (2) 9 nuclear microsatellite markers (n = 146). The overall genetic diversity was moderate, with an indication that different evolutionary forces are at play on a mitochondrial versus nuclear level. The 2 species were delineated based on both marker types, and for R. djiddensis, the sampling locations of South Africa and Mozambique were genetically homogeneous. For R. australiae, significant differentiation was found between sampling locations, with Madagascar and Tanzania being genetically the most similar. This information provides critical insights into the distribution range and population structure of the whitespotted wedgefish species complex that can support the sustainable management of wedgefishes.