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Background: Schistosoma spp. are trematodes occurring in tropical endemic areas but can be imported to non-endemic regions as causes of travel-associated infections. In this study, two pan-trematode-specific real-time PCR assays were evaluated for their diagnostic sensitivity in detecting Schistosoma spp. DNA in diagnostic human samples. Methods: Two previously described pan-trematode-specific real-time PCR assays were comparatively assessed using diagnostic samples containing DNA of either the S. haematobium complex or the S. mansoni complex, as confirmed by Schistosoma species complex-specific real-time PCR. Results: Out of a total of 655 samples containing Schistosoma spp. DNA, positive signals in at least one of the two pan-trematode real-time PCR assays were recorded for 17 (2.6%) nucleic acid extractions. Although sensitivity was in the >90% range for stool samples, only a few individual blood plasma and serum samples, and none of the Schistosoma spp. DNA-containing tissue or urine samples, tested positive by pan-trematode PCR. The lower sensitivity of pan-trematode PCR compared with Schistosoma spp.-specific PCR was semi-quantitatively confirmed by higher cycle threshold (Ct) values in the former. When comparing samples with concordant versus discordant positive results for Schistosoma spp.-specific and pan-trematode PCR, Ct values of the Schistosoma spp.-specific PCR were lower in concordantly positive samples than in discordantly positive samples. Conclusions: While the assessed pan-trematode PCR assays showed insufficient sensitivity as screening tools for blood plasma, blood serum, tissue, and urine samples from individuals with suspected schistosomiasis, they were sufficiently sensitive when applied to stool samples, in which substantial amounts of target DNA, as indicated by low Ct values in the Schistosoma species complex-specific real-time PCR assays, can be expected. For screening for Schistosoma spp. DNA in sample materials other than stool, the use of highly sensitive target-specific PCR remains necessary.