The objective of this study was to show the feasibility to perform Iopamidol-based pH imaging via clinical 3T magnetic resonance imaging (MRI) using chemical exchange saturation transfer (CEST) imaging with pulse train presaturation. The pulse train presaturation scheme of a CEST sequence was investigated for Iopamidol-based pH measurements using a 3T magnetic resonance (MR) scanner. The CEST sequence was applied to eight tubes filled with 100-mM Iopamidol solutions with pH values ranging from 5.6 to 7.0. Calibration curves for pH quantification were determined. The dependence of pH values on the concentration of Iopamidol was investigated. An in vivo measurement was performed in one patient who had undergone a previous contrast-enhanced computed tomography (CT) scan with Iopamidol. The pH values of urine measured with CEST MRI and with a pH meter were compared. In the measured pH range, pH imaging using CEST imaging with pulse train presaturation was possible. Dependence between the pH value and the concentration of Iopamidol was not observed. In the in vivo investigation, the pH values in the human bladder measured by the Iopamidol CEST sequence and in urine were consistent. Our study shows the feasibility of using CEST imaging with Iopamidol for quantitative pH mapping in vitro and in vivo on a 3T MR scanner.