The aim of the study was to assess the presence and distribution of third molars (M3) regarding their autotransplantation in patients with congenital absence of second premolars (PM2). Additionally, M3 development in relation to patients' age and gender was investigated. Panoramic radiographs of non-syndromic patients with at least one congenitally absent PM2 were used to assess the localization and number of missing PM2 and the presence or absence of M3 (minimum age 10 years). The alternate logistic regression model was applied to analyze associations between the presence of PM2 and M3. A total of 131 patients with PM2 agenesis were identified (82 females, 49 males). At least one M3 was present in 75.6% and all M3 were present in 42.7% of patients. A statistically significant association between the number of PM2 and M3 agenesis was found; the effects of age and gender were not significant. More than half of M3 in patients between 14-17 years old had completed ¼ of their root development. The congenital absence of maxillary PM2 was associated with the absence of maxillary PM2, M3, and no correlation was found in the mandible. In patients with PM2 agenesis, at least one M3 is often present and can be considered as a donor tooth for autotransplantation.