This study is motivated by recent investigations in measuring intrinsic and extraneous cognitive load associated with human learning in non-digital environments (Leppink et al. (2013) Development of an instrument for measuring different types of cognitive load. Behave. Res. Methods., 45, 1058–1072; Leppink et al. (2014) Effects of pairs of problems and examples on task performance and different types of cognitive load. Learn. Instr., 30, 32–42) and the increased interest and adoption rates of e-textbooks in higher education. Intrinsic cognitive load associated with the processing of new information is influenced by students’ prior knowledge and learning task difficulty; whereas, extraneous cognitive load involves processes that do not contribute to or even hamper knowledge construction. We adapted Leppink and colleagues’ cognitive load questionnaire to measure intrinsic and extraneous cognitive complexity of e-textbook learning. Undergraduate biology students (N = 1337) completed an online questionnaire, which included e-text cognitive load questions and questions about their preferences and attitudes toward e-textbooks. Results from exploratory and confirmatory factor analyses yielded some support for the use of two constructs of intrinsic and extraneous cognitive load with e-textbook learning. The instrument validity was established using an analysis of the relationships between intrinsic and extraneous cognitive load and student’s academic achievement, e-text preferences and attitude measures. Reading off a screen and navigating and manipulating e-texts were among the factors that negatively correlated with e-text extraneous cognitive load