Nonindigenous species pose a major threat for coastal and estuarine ecosystems. Risk management requires genetic information to establish appropriate management units and infer introduction and dispersal routes. We investigated one of the most successful marine invaders, the ctenophore Mnemiopsis leidyi, and used genotyping by sequencing (GBS) to explore the spatial population structure in its nonindigenous range in the North Sea. We analyzed 140 specimens collected in different environments, including coastal and estuarine areas, and ports along the coast. Single nucleotide polymorphisms (SNPs) were called in approximately 40k GBS loci. Population structure based on the neutral SNP panel was significant (FST 0.02; p <0.01), and a distinct genetic cluster was identified in a port along the Belgian coast (Ostend port; pairwise FST 0.02–0.04; p <0.01). Remarkably, no population structure was detected between geographically distant regions in the North Sea (the Southern part of the North Sea vs. the Kattegat/Skagerrak region), which indicates substantial gene flow at this geographical scale and recent population expansion of nonindigenous M. leidyi. Additionally, seven specimens collected at one location in the indigenous range (Chesapeake Bay, USA) were highly differentiated from the North Sea populations (pairwise FST 0.36–0.39; p <0.01). This study demonstrates the utility of GBS to investigate fine-scale population structure of gelatinous zooplankton species and shows high population connectivity among nonindigenous populations of this recently introduced species in the North Sea.