We herein present the case of a 54-year-old Japanese woman with Whipple disease diagnosed with polymerase chain reaction (PCR) using formalin-fixed paraffin-embedded (FFPE) specimens. The patient complained of weight loss, diarrhea and arthralgia. An endoscopic examination revealed swollen villi in the duodenum and ileum. Pathology demonstrated the presence of numerous macrophages filled with diastase-resistant PAS-positive particles. PCR using FFPE specimens amplified a fragment of 16S rDNA from Tropheryma whipplei. After the administration of ceftriaxone followed by trimethoprim/sulfamethoxazole, no signs of recurrence were observed for two years. The use of FFPE specimens for PCR should be considered for the prompt diagnosis and prevention of disease progression.