Human visceral leishmaniasis (VL) caused by Leishmania infantum in Mediterranean region is still an important public health problem in those countries including Turkey. The asymptomatic presentation of the infection in most of the cases leads to difficulties in the diagnosis and prevention of the spread of infection. The aim of this study was to investigate the seroprevalence of anti-Leishmania antibodies in children inhabiting in the northwestern part of Turkey. A total of 572 healthy children (260 girls, 312 boys) aged between 1-17 years old (mean age: 8.1 years) inhabiting in Eskisehir, Bilecik, Kutahya and Afyon provinces and their counties were included to the study with the informed consent obtained from their families. All serum samples were screened by a commercial ELISA (Leishmania Ab Panel, Cypress Diagnostics, Belgium), two in-house ELISA (with whole antigens and rK39 antigen) tests and an in-house indirect fluorescent antibody test (IFAT). The sera yielding a positive result by commercial ELISA, have been re-evaluated with rK39 dipstick test. Of 572 children, 5.2% (n=30) were found positive with commercial ELISA, 3.8% (n=22) with IFAT, 4.7% (n=27) with in-house whole ELISA, and 3.6% (n=21) with in-house rK39 ELISA. Of 30 commercial ELISA positive sera, 19 (63.3%) gave positive result also by dipstick test. This difference was attributed to the use of multiple antigens belonging to different Leishmania species in commercial ELISA test. The number of children who were seropositive with all of the tests were 15 (2.6%) and with at least three of the tests were 24 (4.2%). Most (76.7%) of the 30 seropositive children with commercial ELISA were found to be inhabiting in Iscehisar town of Afyon province (n=16) and Sogut town of Bilecik province (n=7) where canine VL was also endemic. Our results also indicated that 93.3% (28/30) of seropositive children were living in the rural parts of the study region. Thirty seropositive children were followed-up for one year, but none of them have exhibited clinical findings of VL. As a result, the rate of anti-Leishmania seropositivity (5.2% with at least one test, and 4.2% with at least three tests) in healthy children in the study area should not be ignored, and prevention strategies should be undertaken since our previous study have also showed that the rate of VL in dogs was high and the vector Phlebotomus species were determined in these areas of Turkey.