Background: Recent neurobiological studies have reported that alexithymia may result from altered brain function related to emotional processing. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to regulate central nervous system development associated with psychological processing. We investigated the possibility that polymorphism of the 5-HT transporter-linked promoter region (5-HTTLPR) is associated with alexithymia. Methods: This study included 304 healthy Japanese volunteers (148 males, 156 females). The subjects were categorized according to genotype (L/L, L/S, S/S) and results of the 20-item Toronto Alexithymia Scale (TAS-20), State-Trait Anxiety Inventory (STAI) and Self-Rating Depression Scale (SDS). Results: Subjects with the L/L genotype showed significantly higher TAS-20 scores, as well as significantly higher scores on the difficulty identifying feeling (DIF) subscale, than those with the L/S or S/S genotype (p < 0.05). There was a gender difference in the association between 5-HTTLPR genotype and DIF score. Female subjects with the L/L genotype showed significantly higher DIF scores than those with the L/S or S/S genotype (p ≤ 0.001). Neither STAI nor SDS was significantly associated with the 5-HTTLPR genotype. Conclusion: These results suggest a link between low synaptic 5-HT and alexithymia.