Objective: To explore a novel method for fabricating removable partial dentures (RPDs) using polyetheretherketone (PEEK) by computer-aided design and computer-aided manufacturing (CAD/CAM) technologies and to evaluate their fits for different assemblies in vitro.Methods: A standard stone cast of mandibular partial edentulous jaw was scanned using a lab scanner. Based on the digital cast, thirteen complete RPDs were designed combing dental CAD software and reverse engineering software, and then fabricated using PEEK by milling machine. Fits of assemblies were evaluated quantitatively by measuring the spaces between RPDs and casts. The gaps between RPDs and stone casts in different assemblies were duplicated using light-body silicone impression material and then measured using three-dimensional (3D) digital analysis methods in virtue of a dedicated software. Statistically, one-way analysis of variance (ANOVA) was used to compare the difference of gaps among assemblies in different shapes such as occlusal rest, denture base, and major connector. Paired-samples t test was used to compare the gaps difference for the similar shape assemblies in different areas.Results: One-piece PEEK RPDs were successfully designed and fabricated by CAD/CAM, and all the RPDs were well-seated on stone casts. The gaps between occlusal rests and casts [(84.3±23.6) μm] were significantly larger than those of denture bases [(32.5±27.8) μm] and major connectors [(49.9±47.0) μm], which meant that the fits of denture bases and major connectors were better than that of occlusal rests. However, the fits of all assemblies could be accepted clinically. For the similar shape assemblies in different areas, there were no significantly differences for gaps between distal extension denture bases [(25.1±55.3) μm] and non-extensive denture bases [(41.5±17.7) μm]. The gaps of occlusal rests adjacent and nonadjacent to the edentulous space were (86.1±29.8) μm and (80.8±42.1) μm respectively and there were no significantly difference between them either. These results implied that different locations had no apparent effect on the fits of assemblies.Conclusion: With the computer-aided design and computer-aided manufacturing technology, PEEK can be used to fabricate one-piece removable partial dentures. And all assemblies of the one-piece PEEK RPDs showed satisfying fits in vitro, indicating a promising clinical application in the future.