Purpose To determine whether Acanthamoeba keratitis (AK) patients have higher rates of Acanthamoeba and free-living amoeba (FLA) colonising domestic sinks than control contact lens (CL) wearers, and whether these isolates are genetically similar to the corneal isolates from their CL associated AK. Methods 129 AK patients from Moorefield Eye Hospital, London and 64 control CL wearers from the Institute of Optometry were included in this study. The participants self-collected home kitchen and bathroom samples from tap-spouts, overflows and drains using an instructional kit. The samples were cultured by inoculating onto a non-nutrient agar plate seeded with Escherichia coli, incubated at 32°C and examined for amoebae by microscopy for up to 2 weeks. Partial sequences of mitochondrial cytochrome oxidase genes (coxA) of Acanthamoeba isolates from four AK patients were compared to Acanthamoeba isolated from the patient’s home. The association between sampling sites was analysed with the chi-square test. Results A total of 513 samples from AK patients and 189 from CL controls were collected. The yield of FLA was significantly greater in patients’ bathrooms (72.1%) than CL controls’ bathrooms (53.4%) (p<0.05). Spouts (kitchen 6.7%, bathroom 11%) had the lowest rate of Acanthamoeba isolation compared to drains (kitchen 18.2%, bathroom 27.9%) and overflow (kitchen 39.1%, bathroom 25.9%) either in kitchens or bathrooms (p<0.05). There was no statistically significant difference between the average prevalence of Acanthamoeba in all three sample sites in kitchens (16.9%) compared to all three sample sites in bathrooms (21.5%) and no association for Acanthamoeba prevalence between AK patients and CL controls. All four corneal isolates had the same coxA sequence as at least one domestic water isolate from the patients’ sink of the kitchen and the bathroom. Conclusion The prevalence of Acanthamoeba and FLA was high in UK homes. FLA colonisation was higher in AK patients compared to controls but the prevalence of Acanthamoeba between AK patients and CL controls domestic sinks was similar. This study confirms that domestic water isolates are probably the source of AK infection. Advice about avoiding water contact when using CL’s should be mandatory.