Context Invasive species are major drivers of biodiversity loss, requiring management to reduce their ecological impacts. Population genetics can be applied to delineate management units, providing information that can help plan and improve control strategies. Aim The present study aims to use a genetic approach to test the existence of three previously proposed sambar deer populations in south-eastern Australia. In doing so, the study aims to delineate management units of sambar deer in south-eastern Australia. Methods Sambar deer DNA was sourced opportunistically from tissue samples and targeted scat collection. Samples were collected from three areas in Victoria, south-eastern Australia: Mt Cole (MC), French Island (FI) and eastern Victoria (EV). Contemporary population structure was assessed using a suite of 11 polymorphic microsatellite markers. The number of maternal sambar deer lineages in south-eastern Australia was investigated through sequencing of the mitochondrial (mt)DNA control region. Key results Three distinct genetic clusters were identified. Differentiation among inferred clusters was found to be high, with FST ranging from 0.24 between EV and FI clusters and 0.48 between MC and FI clusters. Two mtDNA haplotypes were identified; R.u1 was found throughout EV and FI, and R.u2 was unique to MC. DNA isolated from scats provided reliable data and proved critical for sampling areas where hunting and culling of deer are not generally undertaken. Conclusions Three genetically distinct sambar deer management units in south-eastern Australia are defined – MC, FI and EV. Sambar deer control strategies should be applied to each management unit independently. This may be difficult or infeasible for the EV management unit, which is large and geographically complex. Further research may help identify additional fine-scale genetic structure in EV, allowing smaller, more practicable management units to be identified. Implications Genetic data can be used to identify management units for invasive species, which will be critical for the development of future management strategies and improving control operations. The approach outlined here could also be applied to improve the management of other introduced deer species in south-eastern Australia.