Folsomia candida (order Collembola), a common soil arthropod, has recently been described living in a shallow (2.5–5.7 m below surface), unconsolidated aquifer in southwestern Michigan. F. candida was haplotyped from 14 wells using inter-simple sequence repeats (ISSRs) markers, and genetic variation in F. candida was used as a bioindicator to identify pore scale to mesoscale migration barriers in the aquifer. With one exception, individual haplotypes were only found in single wells. A significant relationship was identified between interpopulation genetic differentiation and geographic distance, and F. candida exhibits genetic population structuring over a very fine geographic scale (0.65 km2). Monmonier's algorithm was used to identify two most likely subsurface barriers to migration. Hypothesized barriers divided the F. candida populations into three genetically distinct groups, with two groups separated by only 15 m. These groupings are not dependent on groundwater flow direction, and are supported by a minimum spanning network and AMOVA analysis. Because F. candida movement is limited by pore size, these barriers identify local areas of low permeability in the aquifer material. The study suggests that F. candida may serve an important role in identifying geological characteristics and patterns in aquifers that may be difficult to evaluate by other means.