Whipple's disease (WD) is a chronic infection caused by Tropheryma whipplei. A 1-year treatment of oral trimethoprim/sulfamethoxazole (SXT) is commonly used. Advances in the culture of T. whipplei have allowed for full genome sequencing and antibiotic susceptibility testing, which has demonstrated resistance of T. whipplei to trimethoprim. Several mutations in the folP gene that encodes dihydropteroate synthase, the target of sulphonamides, has been reported for one patient with clinically acquired resistance to SXT. Here we report three new patients who experienced clinically acquired resistance to SXT during treatment and one patient with biological failure. Sixty-two folP sequences from DNA samples of 59 WD patients were also obtained. Among the detected amino acid changes, two positions (N4S and S234F) significantly predicted secondary sulfamethoxazole failure (four of five). We suggest that these mutations should be detected at the time of WD diagnosis by sequencing folP in order to avoid sulfamethoxazole monotherapy.