Depression and thermal hypersensitivity share pathogenic features and symptomology, but their pathophysiologic interactions have not been fully eluidated. Dopaminergic systems in the ventrolateral periaqueductal gray and dorsal raphe nucleus have been implicated in these conditions due to their antinociception and antidepression effects, although their specific roles and underlying mechanisms remain obscure. In this study, chronic unpredictable mild stress was used to induce depression-like behaviors and thermal hypersensitivity in C57BL/6J (wild-type) or dopamine transporter promoter (DAT-Cre) mice to establish a mouse model of pain and depression comorbidity. Microinjections of quinpirole, a dopamine D2 receptor agonist, up-regulated D2 receptor expression in dorsal raphe nucleus and reduced depressive behaviors and thermal hypersensitivity with chronic unpredictable mild stress, while dorsal raphe nucleus injections of JNJ-37822681, an antagonist of D2 receptors, had the reciprocal effect on dopamine D2 receptor expression and behaviors. Moreover, using a chemical genetics approach to activate or inhibit dopaminergic neurons in ventrolateral periaqueductal gray ameliorated or exacerbated depression-like behaviors and thermal hypersensitivity, respectively, in dopamine transporter promoter-Cre chronic unpredictable mild stress mice. Collectively these results demonstrated the specific role of ventrolateral periaqueductal gray and dorsal raphe nucleus dopaminergic systems in the regulation of pain and depression comorbidity in mice. PERSPECTIVE: The current study provides insights into the complex mechanisms underlying thermal hypersensitivity induced by depression, and the findings suggest that pharmacological and chemogenetic modulation of dopaminergic systems in the ventrolateral periaqueductal gray and dorsal raphe nucleus may be a promising therapeutic strategy to simultaneously mitigate pain and depression.