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Objectives: The new daily persistent headache (NDPH) is a rare primary headache disorder. However, the underlying mechanisms of NDPH remain incompletely understood. This study aims to apply seed‐based analysis to explore the functional connectivity (FC) of brainstem nuclei in patients with NDPH using resting‐state functional magnetic resonance imaging (MRI). Methods: The FC analysis from the region of interest (ROI) to whole brain voxels was used to investigate 29 patients with NDPH and 37 well‐matched healthy controls (HCs) with 3.0 Tesla MRI. The 76 nuclei in the brainstem atlas were defined as ROIs. Furthermore, we explored the correlations between FC and patients' clinical characteristics and neuropsychological evaluations. Results: Patients with NDPH exhibited reduced FC in multiple brainstem nuclei compared to HCs (including right inferior medullary reticular formation, right mesencephalic reticular formation, bilateral locus coeruleus, bilateral laterodorsal tegmental nucleus‐central gray of the rhombencephalon, median raphe, left medial parabrachial nucleus, periaqueductal gray, and bilateral ventral tegmental area‐parabrachial pigmented nucleus complex) and increased FC in periaqueductal gray. No significant correlations were found between the FC of these brain regions and clinical characteristics or neuropsychological evaluations after Bonferroni correction (p > 0.00016). Conclusions: Our results demonstrated that patients with NDPH have abnormal FC of brainstem nuclei involved in the perception and regulation of pain and emotions. [ABSTRACT FROM AUTHOR]