Although pain is a frequent complaint of patients with chronic kidney disease who undergo hemodialysis, few studies have assessed the functional interference of pain in activities of daily living (ADLs). Hence, the aim of this study was to evaluate the prevalence, location, intensity, and functional interference of pain in ADLs of chronic kidney disease patients undergoing hemodialysis and to estimate the association of specific pain sites with severe functional interference by pain in ADLs. This cross-sectional study included patients with chronic kidney disease undergoing hemodialysis. The prevalence, intensity, and functional interference of pain in ADLs were assessed using the brief pain inventory. Poisson regression was used to calculate the prevalence ratio. A total of 65 patients participated in the study. The overall prevalence of pain was 89.23%; the prevalence of headache was 18.46% and that of pain in the trunk was 55.38%, upper limbs was 35.38%, and lower limbs was 60.00%. The prevalence of moderate and severe pain at the time of hemodialysis was 13.85% and 21.54%, respectively. A high prevalence of severe functional interference of pain in general activity (61.54%), mobility (56.92%), and disposition (55.38%) was observed. Pain is a frequent complaint in patients undergoing hemodialysis, mainly musculoskeletal and intradialytic, and it interferes with ADLs and incapacitates the patient. Pain was highly prevalent in the upper and lower limbs and the trunk. Furthermore, a higher prevalence of severe pain at the time of hemodialysis and functional interference of pain, mainly in general activity, mobility, and disposition, were observed.