Introduction: Dimensional stability of dies is very important in the success of fixed prosthodontics treatments. Impression methods and holding time are the most important factors affecting dimensional stability of the dies. Objectives: This study was performed to investigate the effects of three impression methods and four different dies holding times on the dimensional variations of dies. Material and methods: In this in vitro study, 144 gypsum samples were obtained from an in vitro model in the form of prepared pillars of a long bridge-one pyramid dies, with occlusal diameter of 9.43 mm and height of 12.84 mm, and the other with occlusal diameter of 12.84 mm and height of 12.80 mm. Two dies distance was 44.20 mm. Forty-eight samples were prepared by one-stage molding, 48 samples by two-stage impression with a spacer on the dies and toothless space, and 48 samples by the two-stage impression and with a spacer on the dies. Each of these three groups was divided into four 12-membered subgroups (1 h, 24 h, 48 h, and one week). Dimensional accuracy of the samples was evaluated by a digital caliper and micrometer. To analyze the data, two-way ANOVA test was used (p < 0.05). Results: The mean of diameter, the height of smaller die, and the distance between the two dies was 9.50 ± 0.01, 12.89 ± 0.02, and 44.26 ± 0.01 mm, respectively, in the one-stage impression; 9.46 ± 0.01, 12.87 ± 0.01, and 44.22 ± 0.01 mm, respectively, in the two-stage impression with spacer on the dies; and 9.46 ± 0.01, 12.87 ± 0.01, and 44.22 ± 0.01 mm, respectively, in the two-stage impression with spacer on the dies and toothless space. There was a significant difference between one-stage and two-stage impression methods (p < 0.05), but there was no significant difference between two two-stage impression methods (with different spacer types). Regarding different holding times, no significant difference between them was observed (p > 0.05). Conclusions: The two-stage impression can result in higher dimensional accuracy than the one-stage impression method. Also, when additional silicones are used, the impression can be postponed by one week without significant dimensional changes in the final cast.