Aim Intraspecific genetic variation is a key component of biodiversity, with higher diversity indicating greater resilience and population substructuring suggesting unique evolutionary histories. Comparative approaches, in which intraspecific genetic variation is assessed across multiple species, are powerful tools to identify evolutionary hotspots, but are still rarely applied at spatial scales relevant to conservation planning. Here, we use comparative phylogeography to understand patterns and potential drivers of genetic variation within a biodiversity and ocean warming hotspot. Location The South African coastline, Indian/Atlantic Oceans. Methods A literature search was conducted to obtain mitochondrial DNA cytochrome oxidase c subunit I and cytochrome b sequence data for 17 marine fish and invertebrate species. From these data, we compared averages of haplotype and nucleotide diversity, and within-region ΦST between four biogeographic provinces in the region. Mixed linear models tested whether environmental variability, habitat preference, or geographic location significantly influence genetic variation. Results Average diversity values differed between haplotype and nucleotide diversity, but both broadly displayed highest diversity levels within the South-West bioregion, which is also a region of high levels of within-region ΦST. Range in sea surface temperatures (SSTs) was the only significant fixed-effect term in the haplotype diversity mixed linear models. Mean SST, stability in SSTs since the Mid-Holocene and position within the species' geographic distribution all had no significant effect on genetic variation. Main conclusions Along this coastline characterized by high environmental heterogeneity, we find that variation in temperature is a prominent source of intraspecific variation. Genetic diversity differs between bioregions, but does not display higher levels within the core of each species’ range when assessed across multiple species. With elevated levels of genetic diversity, the South-West region of the South African coast is highlighted as a conservation priority area, representing both high genetic diversity and differentiation across taxa.