Objetivo(s): To evaluate whether oral fluid (OF) and urine can be used as alternative samples to serum to diagnose chikungunya virus (CHIKV) infection by qRT-PCR. Material e Métodos: Serum, OF (collected with a rayon-tipped swab in the sublingual region), and urine samples used in the evaluation were obtained from patients enrolled in a prospective surveillance study to detect arbovirus infection among cases of acute febrile illness (≤7 days of symptom onset). The studied samples belonged to three groups of patients with laboratory evidence of chikungunya: patients with qRT-PCR positive for CHIKV in acute-phase sera (n=19); with CHIKV IgM seroconversion between paired sera (n=12); and with CHIKV IgM detection in acute-phase sera (n=20). The last two groups were negative for CHIKV by qRT-PCR in acute-phase serum. As controls, we used sera, OF, and urine from patients with other acute febrile illnesses (n=28) and from patients with RT-PCR-confirmed dengue (n=16). All samples underwent RNA extraction (Maxwell®, Promega, Madison, USA), followed by CHIKV qRT-PCR (CDC-TRIOPLEX). CHIKV IgM antibodies were investigated using IgM ELISA kits (InBios, Seattle, USA). Resultados e Conclusão: Of the 19 patients with qRT-PCR positive for CHIKV in sera, 9 (47.4%) and one (5.2%) were also positive by qRT-PCR in OF and urine, respectively. Of the 12 patients with evidence of CHIKV infection solely based on CHIKV IgM seroconversion, 2 (16.7%) were positive for CHIKV by qRT-PCR in OF, and none in urine. None of the 20 patients belonging to the group with CHIKV IgM antibodies in acute-phase sera were positive for CHIKV by qRT-PCR in OF and urine. None of the samples from the control groups were positive (specificity: 100%).Although the CHIKV qRT-PCR in OF was positive in some patients with CHIKV IgM seroconversion who were negative by qRT-PCR in sera, the qRT-PCR sensitivity in OF was about 50% among patients who had a positive CHIKV qRT-PCR in serum. Thus, while OF may be useful as an alternative non-invasive specimen to diagnose acute chikungunya, a negative result cannot rule out an infection. The frequency at which acute-phase urine showed detectable levels of CHIKV RNA in patients with detectable CHIKV RNA in sera was too low to suggest the use of urine as an alternative acute-phase sample. Further research is needed to evaluate whether OF and urine collected later in the disease course may be helpful in diagnosing chikungunya.