The rK39 rapid immunochromatographic test (ICT) is now being widely used in the diagnosis of visceral leishmaniasis (VL) using serum. We evaluated the presence of anti-rK-39 antibody in human saliva being noninvasive to replace the invasive procedures of diagnosis. Enzyme-linked immunosorbent assay (ELISA) and ICT assays were performed in 300 subjects: 114-confirmed VL patients, 95 and 47 healthy controls from endemic and nonendemic regions, respectively, and 44 subjects with different diseases. Sensitivity in saliva was 83.3% by ELISA and 82.5% by ICT, compared with 100% for both ICT and ELISA in serum. Specificity in saliva was 100%, 90.5%, and 88.6% with ELISA, and 91.48%, 91.57%, and 84.06% using ICT, in nonendemic, endemic, and different diseases, respectively. In serum, specificity was 97%, 88.5%, and 89% by ELISA and 100%, 94.7%, and 95.5% by ICT in nonendemic, endemic, and different diseases, respectively. Saliva is not suitable for diagnosis of VL because of low sensitivity.