Early life stress disrupts development of the human and animal brain and increases the risk of psychophysiological disorders and susceptibility to addiction in adulthood. Maternal separation (MS), an animal model of early life stress, was shown to raise predisposition to addictive behaviours and change neuronal activity as well as dendritic spine density in a range of brain structures. Mesocorticolimbic dopaminergic pathways originating from the ventral tegmental area (VTA) play a crucial role in the development of addiction, however, the influence of MS on VTA neuronal architecture remains obscure. AIM(S): The current study aimed to verify the influence of MS on VTA neuronal dendritic spine density, a possible anatomical substrate of functional changes in the ascending dopaminergic pathways. METHOD(S): Female rat pups were separated from dams for 3 hours daily from PND2 to 14. At PND65, rats were decapitated, Golgi-Cox staining was performed, and the density of spines was calculated manually on I‑III‑ order dendrites. Given the functional and anatomical heterogeneity of the VTA, analyzed neurons were assigned to specified VTA sub‑regions. RESULTS: In rats subjected to MS, significantly lower density of dendritic spines on neurons in ventromedial (II‑order branches – 15%), dorsolateral (II‑ and III‑order segments – 24 – 23% respectively), and dorsomedial (I‑order branches – 25%) VTA, was observed when compared to control. No significant changes in spine density were observed in ventrolateral VTA (only 6% decrease in spine density). CONCLUSIONS: The observed decrease in dendritic spine density in VTA neurons can be linked to a reduced number of excitatory synapses that may underlie altered activity of mesocorticolimbic pathways, altered dopamine release, and increased susceptibility to addictions observed after MS and childhood trauma. Sub‑region specificity of observed changes points to varied sensitivity of VTA neurons to stress. FINANCIAL SUPPORT: Funding: NSC-Poland UMO‑2016/21/B/NZ4/00204.