Pelagia noctiluca is thought to have a global distribution, yet our understanding of genetic connectivity across the range of this problem animal is poor. Here, we investigate the genetic structure of populations off southern Africa using mitochondrial COI and nuclear ITS1 and ITS2 genes, and compare the results to recent work conducted in the North-East Atlantic. Analyses showed significant differentiation between the southern and northern Atlantic population groups (COI Φ st = 0.72, ITS2 Φ st = 0.23, p < 0.001), which suggests historical rather than contemporary gene flow. Southern African samples showed high haplotypic (h = 1) and low nucleotide (π = 0.008) diversity, similar to those from Europe. Phylogenetic analyses suggest South African samples to have diverged earlier than those from the northern Atlantic.