This study involved the development and application of a two-tier diagnostic test for measuring students' understanding of cell division and reproduction. The instrument development procedure had three general steps: defining the content boundaries of the test, collecting information on students' misconceptions, and instrument development. Misconception data were collected from interviews and multiple-choice questions with open response. The data were used to develop fourteen two-tier multiple-choice items. The conceptual knowledge examined was preliminary functions of cell division, basic processes in cell division, mitotic cell cycle and asexual reproduction, the role of meiosis in sexual life cycles, as well as cell types and cell division. The diagnostic instrument was administered to 403 high-school students. The reliability coefficient of the test was 0.81. Difficulty indices ranged from 0.31 to 0.72, and discrimination indices ranged from 0.23 to 0.61. Results of the cell division and reproduction diagnostic test suggested that students did not acquire a satisfactory understanding of cell division and reproduction concepts. The obtained misconceptions and the reasons for them were discussed in the analysis of the items which could inform biology instruction and resource.