Acanthamoeba is one of the most common free-living amoebas (FLA) that present in environment. In humans, Acanthamoeba can cause an infection of the eye termed Acanthamoeba keratitis, which mostly occurs in contact lens wearers. In the present study, we aimed to screen the presence of Acanthamoeba DNA in stray dogs using previously collected conjunctival swab samples in a hyper-endemic area for canine leishmaniasis. Totally, 184 dogs were included in the study and 27 of them (14.6%) were found positive for Acanthamoeba according to the 18s rRNA gene sequencing. Two different genotypes (T4 and T5) were identified and T5 was firstly reported in Turkey in the present study. Statistical analysis was performed and no correlation was found between Leishmania and Acanthamoeba positivity (P < 0.05). To best of our knowledge, this is the first study conducted to screen Acanthamoeba among stray dogs. Further studies are necessary to reveal the infection status and genotypes among dogs and its possible correlation with leishmaniasis. (C) 2016 Elsevier B.V. All rights reserved.