Objective This study compared the marginal and internal fit of implant-supported fixed dentures fabricated using CAD/CAM systems. Materials and methods A lower jaw model representing partial edentulism was produced. Two dental implants were inserted in the area of teeth 35 and 37, onto which cemented abutments were screwed. The model was scanned using a laboratory scanner and transferred to a design software program for substructure fabrication. Sixty substructures were fabricated out of each group for six substructure types (n = 10), cast Co-Cr (control), milling Co-Cr, laser sintering Co-Cr, titanium (Ti), zirconium, and polyetheretherketone (PEEK) substructures. The marginal and internal fit was evaluated using a silicone replica viewed under a stereomicroscope. The data were analyzed using the statistical package program for social sciences (SPSS, Chicago, IL, USA, v. 17) at a significance level of 0.05. Marginal and internal gaps were compared using the one-way ANOVA test and Tukey's post hoc test. The differences between abutment teeth were determined using the independent sample t-test. Results There was a significant difference in the marginal gap between PEEK and Ti groups (p < 0.05) but no difference between other groups (p > 0.05). There was a significant difference in the internal gap between PEEK, laser sintering Co-Cr, and milling Co-Cr groups (p < 0.05) but no difference between other groups (p > 0.05). The PEEK group had a higher marginal gap than the Ti group and a higher internal gap than the DMLS Co-Cr group (p < 0.05). Conclusion All substructures have a marginal and internal fit within acceptable clinical limits.