Living kidney donors (LKDs) may feel more anxious about kidney failure now that they have only one kidney and the security of a second kidney is gone. The aim of this cross-sectional study was to develop and empirically validate a self-report scale for assessing fear of kidney failure in former LKDs. Participants were 364 former LKDs within the past 10 years at five US transplant centers and 219 healthy nondonor controls recruited through Mechanical Turk who completed several questionnaires. Analyses revealed a unidimensional factor structure, excellent internal consistency (α = 0.88), and good convergent validity for the Fear of Kidney Failure questionnaire. Only 13% of former donors reported moderate to high fear of kidney failure. Nonwhite race (OR = 2.9, P = 0.01), genetic relationship with the recipient (OR = 2.46, P = 0.04), and low satisfaction with the donation experience (OR = 0.49, P = 0.002) were significant predictors of higher fear of kidney failure. We conclude that while mild anxiety about kidney failure is common, high anxiety about future renal failure among former LKDs is uncommon. The Fear of Kidney Failure questionnaire is reliable, valid, and easy to use in the clinical setting.