Acanthamoeba keratitis is a disease affecting the cornea caused by unicellular protozoa Acanthamoeba spp. that live freely in nature. This study aims to determine the potential for transmission and genetic characteristics of Acanthamoeba spp. from contact lens cleaning solutions and household water sources for contact lens users. The was a cross-sectional study that was conducted in January-May 2019. The collected samples were contact lens and tap water from 53 lens users in Jakarta. Acanthamoeba spp was detected microscopically from culture and molecularly by PCR-sequencing. From 53 contact lenses samples, there were 2 positive samples of Acanthamoeba sp and three positive samples of other free-living amoebae. From the culture of 53 tap water samples, there were 5 positive cultures of Acanthamoeba spp. and 34 positive cultures of free-living amoeba. From the positive PCR results on contact lenses and tap water, it was found that there was a similarity in 1 sample of contact lenses and tap water from 1 subject. Sequencing analysis found that only 1 contact lens sample was Acanthamoeba castellani genotype T4. Meanwhile, from the tap water sample, there were 5 samples that could be sequenced. The sequencing analysis results showed that 2 samples were Acanthamoeba lenticulata genotype T5 and 3 were Acanthamoeba jacobsi genotype T15.