The thalamus is a key region for the transmission of nociceptive information in the central modulation of pain and has been studied in the setting of numerous chronic pain conditions. Brain-derived neurotrophic factor (BDNF) is considered an important modulator for mediating nociceptive pathways in chronic pain. The present study aimed to investigate whether there was thalamus-related abnormal functional connectivity or relevant serum BDNF level alterations during periovulation in long-term primary dysmenorrhea (PDM). Thalamic subregions were defined according to the Human Brainnetome Atlas. Functional connectivity analyses were performed in 36 patients in the periovulatory phase and 29 age-, education-, and gender-matched healthy controls. Serum BDNF levels were evaluated by enzyme-linked immunosorbent assay and a significantly higher BDNF level was detected in PDM patients. Compared with HCs, PDM patients had abnormal functional connectivity of thalamic-subregions, mainly involving with prefrontal cortex, sensorimotor cortex, and temporal cortex. In addition, the functional connectivity of thalamic-subregions showed significant interactive effect correlated with serum BDNF level between PDM and HCs. It has been suggested that there were maladaptive or adoptive alteration associated with chronic menstrual pain even without the ongoing menstrual pain. BDNF might play a role in the development and chronicity of central nervous system dysfunction. These findings provided more accurate information about the involvement of the thalamus in the pathophysiology of PDM.