This study investigated undergraduate students' understanding of radiation and radioactivity. Totally 682 undergraduate students were participated in this study. The undergraduates were selected from science teaching department, chemistry department, and physics department from a university. Undergraduate students' understandings were identified by using a two-tier diagnostic test consisting 20 items. A one-way analysis of variance and Bonferroni test were conducted to compare the scores. The results showed that there was significant difference between all groups in terms of mean scores. Based on the results, it was found that undergraduate students attending in science teaching department had significantly higher scores then undergraduates from chemistry and physics department. According to the results, 32 misconceptions related to ionizing radiation, non-ionizing radiation, nuclear and chemical reactions, half life, types of radioactive radiation, types of radiation, nuclear and chemical reactions, nuclear forces, fission and fusion, radiation in daily life were identified.