Background Patient-specific instrumentation in TKA has the proposed benefits of improving coronal and sagittal alignment and rotation of the components. In contrast, the literature is inconsistent if the use of patient-specific instrumentation improves alignment in comparison to conventional instrumentation. Depending on the manufacturer, patient-specific instrumentation is based on either MRI or CT scans. However, it is unknown whether one patient-specific instrumentation approach is more accurate than the other and if there is a potential benefit in terms of reduction of duration of surgery. Questions/purposes We compared the accuracy of MRIand CT-based patient-specific instrumentation with conventional instrumentation and with each other in TKAs. The three approaches also were compared with respect to validated outcomes scores and duration of surgery. Methods A randomized clinical trial was conducted in which 90 patients were enrolled and divided into three groups: CT-based, MRI-based patient-specific instrumentation, and conventional instrumentation. The groups were not different regarding age, male/female sex distribution, and BMI. In all groups, coronal and sagittal alignments were measured on postoperative standing long-leg and lateral radiographs. Component rotation was measured on CT scans. Clinical outcomes (Knee Society and WOMAC scores) were evaluated preoperatively and at a mean of 3 months postoperatively and the duration of surgery was analyzed for each patient. MRI-and CT-based patientspecific instrumentation groups were first compared with conventional instrumentation, the patient-specific instrumentation groups were compared with each other, and all three approaches were compared for clinical outcome measures and duration of surgery. Results Compared with conventional instrumentation MRI-and CT-based patient-specific instrumentation showed higher accuracy regarding the coronal limb axis