The aim of the present in vitro study was to evaluate the influence of different processing routes on the fitting accuracy of four-unit zirconia fixed dental prostheses (FDPs) fabricated by computer-aided design/computer-aided manufacturing (CAD/CAM). Three groups of zirconia frameworks with ten specimens each were fabricated. Frameworks of one group (CerconCAM) were produced by means of a laboratory CAM-only system. The other frameworks were made with different CAD/CAM systems; on the one hand by in-laboratory production (CerconCAD/CAM) and on the other hand by centralized production in a milling center (Compartis) after forwarding geometrical data. Frameworks were then veneered with the recommended ceramics, and marginal accuracy was determined using a replica technique. Horizontal marginal discrepancy, vertical marginal discrepancy, absolute marginal discrepancy, and marginal gap were evaluated. Statistical analyses were performed by one-way analysis of variance (ANOVA), with the level of significance chosen at 0.05. Mean horizontal discrepancies ranged between 22 μm (CerconCAM) and 58 μm (Compartis), vertical discrepancies ranged between 63 μm (CerconCAD/CAM) and 162 μm (CerconCAM), and absolute marginal discrepancies ranged between 94 μm (CerconCAD/CAM) and 181 μm (CerconCAM). The marginal gap varied between 72 μm (CerconCAD/CAM) and 112 μm (CerconCAM, Compartis). Statistical analysis revealed that, with all measurements, the marginal accuracy of the zirconia FDPs was significantly influenced by the processing route used (p < 0.05). Within the limitations of this study, all restorations showed a clinically acceptable marginal accuracy; however, the results suggest that the CAD/CAM systems are more precise than the CAM-only system for the manufacture of four-unit FDPs.