Background and Aim: Occlusion analysis in the primary dentition period can predict the occlusion of permanent dentition. This study aimed to assess the frequency of malocclusion in primary dentition period in 3 to 5-year-old preschoolers in Isfahan city, Iran. Materials and Methods: This descriptive, cross-sectional study analyzed the occlusion of primary teeth in 400 children between 3 to 5 years who were selected from 8 kindergartens in Isfahan city during 2019-2020. The dentition of children was clinically examined by a dental mirror and a tongue blade after obtaining consent from the parents. The children had all the primary teeth with no permanent tooth, and had no extraction in their treatment plan. The primary molar relationship, canine relationship, and anterior or posterior crossbite, overbite and overjet were all assessed and recorded. Data were analyzed using the Chi-square, Kruskal-Wallis, and Fisher's exact tests. Results: Of all, 82.75% of the children had flush terminal plane (FTP), 10.25% had distal step, and 6% had mesial step. Class I canine relationship had the highest prevalence (87%) followed by class II (8.25%) and class III (4%). The frequency of increased overjet was 14.25%. Deep bite (15%) was the most prevalent malocclusion. Anterior and posterior crossbite had 6.25% prevalence. Conclusion:Relatively high prevalence of malocclusion in our study population calls for in-time assessment of primary occlusion for timely detection and management of malocclusions in the primary dentition period to prevent their aggravation.