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This study was motivated by recent investigations in measuring intrinsic and extraneous cognitive load associated with human learning in non-digital environments (Leppink et al., 2013; 2014) and the increased interest and adoption rates of e-textbooks in higher education. We adapted Leppink and colleagues’ cognitive load questionnaire to measure intrinsic and extraneous cognitive complexity of e-textbook learning. 1,337 undergraduate biology students rated their preferences and attitudes toward e-textbooks versus printed learning materials and completed the e-textbook cognitive load questionnaire. The data supported a two-factor pattern for intrinsic and extraneous cognitive load associated 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 attitudes measures.