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Tree thinking refers to an approach to evolution education that emphasizes reading and interpreting phylogenetic trees. We studied the relationship between introductory biology students’ tree-thinking ability and their acceptance of evolutionary theory. Comparisons between a semester in which interpretation of phylogenetic trees and related concepts were taught as stand-alone topics versus one in which tree thinking was used as an organizing framework for the course curriculum found significant increases in students’ ability to read and interpret trees in both semesters, but only in the tree thinking semester was there a significant increase in students’ scores on the Measure of the Acceptance of the Theory of Evolution (MATE) instrument. Over four additional semesters, the Tree Thinking Concept Inventory (TTCI) was used to assess students’ tree-thinking abilities before and after the course. We found that MATE posttest scores correlated with gains in tree-thinking ability. These increases in MATE posttest scores were predominantly due to students’ reporting greater acceptance of the scientific evidence supporting evolutionary theory. Increased acceptance of evolution in a tree-thinking versus non-tree-thinking semester and significant positive correlations between TTCI and MATE scores indicate there is a relationship between tree-thinking ability and acceptance of evolution. This result suggests that, as a framework to connect different forms of data and investigate a variety of biological phenomena, tree thinking can promote greater acceptance of the evidence and scientific validity of evolutionary theory in introductory biology students.