Objectives Feline herpesvirus-1 (FHV-1), feline calicivirus (FCV) and Chlamydia felis are involved in feline upper respiratory tract disease (FURTD). Clinical signs caused by these agents can overlap, and the involvement of certain pathogens is often unpredictable. The objectives of this study were to compare detection rates of FHV-1, FCV and C felis at different sampling sites, and to investigate the correlation between positive test results and clinical signs in cats with FURTD. Methods Swabs were taken from the nose, pharynx, tongue and conjunctiva of 104 cats with signs of FURTD. Real-time PCR was performed on all samples for the detection of FHV-1, FCV and C felis. Results Infectious agents were identified in 93 (89.4%) cats. Of these, 55.8% were positive for FHV-1, 50.0% for FCV and 35.6% for C felis. FCV was found more frequently in the oropharynx (92.3% of FCV-positive cats) and on the tongue (90.4%) than the conjunctiva (38.5%) (P <0.001). There was no significant difference between the four sampling sites for the detection of FHV-1 and C felis. If nasal samples had also been taken, 94.9% of FHV-1-positive cats, 96.2% of FCV-positive cats and 81.1% of C felis-positive cats would have been detected. Conclusions and relevance The oropharynx can be recommended as the preferred single sampling site for the detection of FCV, FHV-1 and C felis if only one sample can be taken; however, taking samples at different sites significantly increases the detection rate for all pathogens studied. Interestingly, sampling from a site with FURTD-associated lesions did not increase the likelihood of detecting the infectious agents