Although there is growing evidence that an increase in mucosal mast cells (MMCs) in the small and large intestine is associated with visceral hypersensitivity, few studies have evaluated MMCs in humans with esophageal symptoms. The aim of this study was to investigate esophageal MMC distribution in patients with non-cardiac chest pain (NCCP) and to examine the association between the number of gut MMCs and other functional gastrointestinal disorders. Forty-two consecutive NCCP patients and 10 healthy controls completed a questionnaire for bowel symptoms, chest pain intensity score, and psychologic depression. Esophageal, duodenal, and rectal MMCs were identified immunohistochemically and quantified by image analysis. Numbers of MMCs were significantly higher in NCCP patients vs healthy controls (11.8 ± 5.6 vs 7.6 ± 3.7 MMCs/high-power field, p = 0.026). In comparison of subgroups classified by 24-h impedance-pH monitoring, esophageal MMC counts were highest in the hypersensitive esophagus group (p < 0.01) and were also significantly increased in the functional chest pain group (p < 0.05). A positive correlation between esophageal and duodenal MMC counts was observed in patients with functional dyspepsia (FD; Spearman ρ = 0.604, p = 0.037). In particular, patients with clinical overlap with irritable bowel syndrome showed a strong positive correlation between esophageal and rectal MMC numbers (Spearman ρ = 0.857, p = 0.010). Among NCCP patients, increased MMC infiltration occurs in subgroups with hypersensitive esophagus and functional chest pain. In subpopulations with overlap with FD or irritable bowel syndrome, esophageal MMC counts demonstrated significant positive correlations with duodenal or rectal MMC counts.