Genotype data for 399 squirrel gliders (Petaurus norfolcensis) at 8 micro satellite loci. The data support the publication "Evaluating the success of wildlife crossing structures using genetic approaches and an experimental design: lessons from a gliding mammal", Journal of Applied Ecology, 2017 (DOI to follow). Study was conducted along the Hume Highway in south-east Australia, 2005-2013. Data are provided for 13 sites divided into 4 treatment types, before and after crossing structures were added to a highway. Genotypes were first grouped into genetic regions (southern, central and northern) and then population structure within each region (among sites, sides of the highway, and time periods) were investigated. Contact Kylie Soanes for further information (ksoanes@unimelb.edu.au). Microsatellite loci are described in Millis 2000 (Molecular Ecology) and Soanes et al 2014 (Conservation Genetic Resources).