Magnetic resonance imaging with arterial spin labeling (ASL) is a noninvasive approach to measure organ perfusion. The purpose of this study was to evaluate the reproducibility of ASL kidney perfusion measurements with semiautomatic segmentation, which allows separate quantification of cortical and medullary perfusion.The right kidneys of 14 healthy volunteers were examined 6 times on 2 occasions (3 times at each occasion). There was a 10-minute pause between each examination and a 14-day interval between the 2 occasions. Cortical, medullary, and whole kidney parenchymal perfusion was determined with customized semiautomatic segmentation software. Coefficient of variances (CVs) and intraclass correlations (ICCs) were calculated.Mean whole, cortical, and medullary kidney perfusion was 307.26 ± 25.65, 337.10 ± 34.83, and 279.61 ± 26.73 mL/min/100 g, respectively. On session 1, mean perfusion for the whole kidney, cortex, and medulla was 307.08 ± 26.91, 336.79 ± 36.54, and 279.60 ± 27.81 mL/min/100 g, respectively, and on session 2, 307.45 ± 24.65, 337.41 ± 33.48, and 279.61 ± 25.94 mL/min/100 g, respectively (P > 0.05; R2 = 0.60/0.59/0.54). For whole, cortical, and medullary kidney perfusion, the total ICC/CV were 0.97/3.43 ± 0.86%, 0.97/4.19 ± 1.33%, and 0.96/4.12 ± 1.36%, respectively. Measurements did not differ significantly and showed a very good correlation (P > 0.05; R2 = 0.75/0.76/0.65).ASL kidney measurements combined with operator-independent semiautomatic segmentation revealed high correlation and low variance of cortical, medullary, and whole kidney perfusion.