Phylogenetic tree diagrams are commonly found in introductory biology curricula and represent the evolutionary relationships of organisms. Tree-thinking, or the ability to accurately interpret, use, and generate these phylogenetic representations, involves a challenging set of skills for students to learn. Although many introductory biology courses incorporate tree-thinking instruction, few studies have identified which instructional methods provide the best learning gains for students. We gathered data from 884 introductory biology students using the Basic Evolutionary Tree-Thinking Skills Inventory (BETTSI) to measure tree-thinking learning gains. We measured tree-thinking differences across five sections of introductory biology, each offering a different instructional intervention, and compared differences among STEM majors and non-STEM majors. After calculating paired differences, we performed a two-way repeated measure analysis of variance (ANOVA) and Scheffe's post hoc test to identify significant differences among and between the different interventions. We found that students who engaged in active tree-thinking instruction had significantly higher tree-thinking learning gains than students who participated in passive or no instruction. Furthermore, these learning gains became even more significant as active-learning became more multifaceted. These active-learning approaches also removed knowledge gaps between STEM majors and non-majors. Instructors must select explicit and active pedagogical approaches to support student tree-thinking to accomplish positive learning gains for all students.