This study involved the development and application of a two-tier diagnostic test measuring students' understanding of flowering plant growth and development. 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 answers. The data were used to develop 13 two-tier multiple-choice items. The conceptual knowledge examined was flowering plant life cycles, reproduction, precondition of germination, plant nutrition, and mechanism for growth and development. The diagnostic instrument was administered to 477 high school students. The correlation coefficient of test-retest was 0.75. Difficulty indices ranged from 0.24 to 0.82, and discrimination indices ranged from 0.32 to 0.65. Results of the Flowering Plant Growth and Development Diagnostic Test suggested that students did not acquire a satisfactory understanding of plant growth and development concepts. Nineteen misconceptions were identified through analysis of the items that could inform biology instruction and resource.