Students have difficulties understanding the crossing-over field concept in genetic courses because within the concept of crossing-over there are facts and problems that are difficult to solve as well as the absence of valid, practical, and effective student worksheets. So that the learning process is complete, activities and student learning outcomes become low. The purpose of this research is to develop a student worksheet based on mastery learning that is valid, practical, and effective on crossing-over field concept. The type of research is developed using 4-D model. The data analysis technique is a descriptive analysis that describes the results of validity, practicality, and effectiveness of the student worksheet. The result of the validity test and the practicality of student worksheet based on mastery learning on crossing-over field concept is a validity value of 88% and practical value of 80,24%. This student worksheet is used because it can increase student activity and learning outcomes.