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Background . This study aimed to evaluate the effect of different pouring times and spacer thicknesses on the three-dimensional accuracy of casts made of 3D-printed custom trays. Methods. A partial edentulous maxillary model was scanned for fabricating custom acrylic trays. Twenty custom trays were created using a CAD/CAM system and divided into two groups in terms of their spacer thicknesses (2 mm and 4 mm). All the trays were designed with 2-mm thickness, multiple retentive holes measuring 2 mm in diameter, and three interior seating stops (two on the edentulous ridge and one on the incisal edge of the central incisors). Impressions were made using monophasic polyvinyl siloxane and poured in two different times (one hour and 24 hours after removal) with type IV dental stone. All the casts were scanned to measure three distances (inter-buccal cusps, inter-palatal cusps, and inter-fossa distances) between the two first premolars. The data were analyzed with two-way ANOVA and Bonferroni test at a significance level of 0.05. Results. There was no significant difference between the 3D accuracy of casts made using two different spacer thicknesses poured at 1-hour and 24-hour intervals. However, there was a difference between casts made after 1 hour and 24 hours when using custom trays with 2 mm of spacer thickness in terms of inter-buccal distance. Conclusion. There was no significant difference between the accuracy of casts made using custom trays with either 2 or 4 mm of spacer thickness, which were poured 1 hour or 24 hours after tray removal.