Differentiation between Whipple disease (WD) patients and patients carrying Tropheryma whipplei but suffering from disease other than WD (“carriers”) remains complex. We aimed to evaluate T. whipplei PCR among patients with WD and carriers in a large cohort at our referral clinical microbiology laboratory. This is an observational retrospective cohort study, including all patients between 2008 and 2020 with at least one positive result for T. whipplei using the real-time PCR RealCycler TRWH-UX kit. A total of 233 patients were included: 197 were considered carriers, and 36 had WD.