Plant tissue culture can be used for plant propagation or for biotechnology purposes, one of which is anther tissue culture. Anther culture of chili plants is difficult because of callus growth, regeneration of plant organs, and low effectiveness. Pretreatment such as cold temperatures can increase the induction of callus growth and embryogenesis in chili anther cultures. However, the chili anther culture process is influenced by many factors such as plant varieties or genotypes, stages of microspore development, type of media and concentration of PGR used, pretreatment given, and incubation environment. This study aimed to determine the effect of cold pretreatment period on callus formation of anther culture of local Aceh red chili plants, the Perintis genotype. This research was carried out at the Plant Tissue Culture Laboratory, Department of Agrotechnology, Faculty of Agriculture, Syiah Kuala University, from December 2021 to April 2022. This study used a completely randomized design (CRD) with a non-factorial pattern with 4 treatments, namely control, cold pretreatment 4℃ for 24 hours, 48 hours, and 72 hours, each treatment was repeated 6 times with a total of 60 anthers in each treatment. This study used MS media with the addition of a concentration of 2 mgL-1 IAA + 0.2 mgL-1 Kinetin. Parameters observed were microspore development stage, callus growth period, percentage of callus growth, percentage of contamination, type of contamination, percentage of embryogenesis, and callus color. The results showed that the duration of cold pretreatment had no significant effect on the callus formation of the anther culture of Perintis red chili plants. The results showed that the 6-day-old Perintis genotype red chili flower contained microspores at the binucleat pollen. The highest percentage of anther culture callus growth was found in the control, which was 8.3%. The percentage of callus growth in 48 hours and 72 hours cold pretreated anther obtained 6.6%, respectively. Meanwhile, the lowest percentage of anther culture callus growth was obtained from the 24-hour cold pretreatment at 5%. The lowest percentage of contamination was obtained from the 48-hour cold pretreatment of 8.3%. Bacteria is the most common contaminant causing contamination in this study at 7.9%. The highest percentage of embryogenesis was obtained from 72 hours of cold pretreatment at 3.3%. The callus produced by anthers was white (11.6%), yellowish white (10%), and brownish white (5%).