AbstractAlcohol use disorders represent an extensive socioeconomic burden, yet effective treatment options are suboptimal. A major hurdle in treating alcohol use disorders is the high rate of relapse. Stress is a major factor that promotes relapse in abstinent drug users; therefore, understanding neural mechanisms that underpin the effects of stress on alcohol seeking is critical. In rodent models of stress‐induced relapse, the α2‐adrenoceptor antagonist, yohimbine, is a widely used chemical stressor to elicit reinstatement of drug/alcohol seeking. However, the exact mechanism how yohimbine precipitates reinstatement of alcohol seeking and the pattern of neural activation associated with yohimbine‐induced reinstatement is poorly understood. Therefore, we counted Fos‐protein positive nuclei across 42 brain regions in alcohol‐experienced alcohol preferring rats that received either yohimbine in the home‐cage (1 mg/kg i.p.) or following yohimbine‐induced reinstatement of alcohol seeking. The number of Fos‐protein positive nuclei was increased in the prefrontal cortex and extended amygdala after home‐cage yohimbine compared to naïve‐ and vehicle‐treated rats. Yohimbine‐induced reinstatement increased the number of Fos‐protein expressing nuclei in multiple other regions including the thalamus, hypothalamus and hippocampus. We then examined inter‐regional correlations in Fos‐protein expression for all 42 brain regions, which showed Fos expression was more strongly positively correlated following yohimbine‐induced reinstatement of alcohol seeking, compared to home‐cage yohimbine. These data suggest low‐dose yohimbine in a non‐drug‐associated context activates stress/impulsivity centres within the brain, whereas yohimbine in the drug‐associated context recruits additional brain regions to drive alcohol seeking.