Little is known about the spatial and temporal scales at which planktonic organisms are genetically structured. A previous study of mitochondrial DNA (mtDNA) in the holoplanktonic chaetognath Sagitta setosa revealed strong phylogeographic structuring suggesting that Northeast (NE) Atlantic, Mediterranean and Black Sea populations are genetically disjunct. The present study used a higher sampling intensity and a combination of mitochondrial and four microsatellite markers to reveal population structuring between and within basins. Between basins, both marker sets indicated significant differentiation confirming earlier results that gene flow is probably absent between the respective S. setosa populations. At the within-basin scale, we found no evidence of spatial or temporal structuring within the NE Atlantic. In the Mediterranean basin, both marker sets indicated significant structuring, but only the mtDNA data indicated a sharp genetic division between Adriatic and all other Mediterranean populations. Data were inconclusive about population structuring in the Black Sea. The levels of differentiation indicated by the two marker sets differed substantially, with far less pronounced structure detected by microsatellite than mtDNA data. This study also uncovered the presence of highly divergent mitochondrial lineages that were discordant with morphology, geography and nuclear DNA. We thus propose the hypothesis that highly divergent mitochondrial lineages may be present within interbreeding S. setosa populations.