The lucerne flea, Sminthurus viridis (Collembola: Sminthuridae) (L.) is a major pest of broadacre agriculture across southern Australia. Few molecular studies have been conducted on S. viridis and none have examined its population genetics, despite the importance for developing effective control strategies. Here, we characterize the genetic structure of Australian populations using three allozyme and eight microsatellite loci, as well as sequencing a fragment of the mitochondrial DNA cytochrome oxidase I gene. We found that S. viridis in Australia are diploid, sexually reproducing and exhibit significant population structure as a result of limited gene flow. Despite significant differentiation between populations, there was very low cytochrome oxidase subunit I (COI) gene sequence variation, indicating the presence of a single species in Australia. The observed structure only marginally complied with an ‘isolation by distance’ model with human-mediated long-distance dispersal likely occurring. Allozymes and microsatellites gave very similar FST estimates, although differences found for novel alternative estimates of differentiation suggest that the allozymes did not capture the full extent of the population structure. These results highlight that control strategies may need to vary for locally adapted S. viridis populations and strategies aimed at limiting the spread of any future pesticide resistance will need to manage the effects of human-mediated dispersal.