Advances in neuro-technology provide new insights into how individual students learn in educational contexts. However, applying it poses challenges for teachers in natural settings. This paper presents an example of the use and applicability of eye-tracking technology in Higher Education. We worked with a sample of 20 students from three universities (Burgos and Valladolid in Spain and Miño in Portugal). The objectives were: (1) to determine whether there were significant differences in indicators of cognitive effort (FC, FD, SC, PD, VC) found with eye-tracking technology between students with and without prior knowledge; (2) to determine whether there were clusters of learning behavior patterns among students; and (3) to analyze differences in the visualization of behavior patterns. A quasi-experimental design without a control group and a descriptive design were used. The results indicated significant differences in learning outcomes between students with and without prior knowledge. In addition, two clusters were found in indicators of cognitive effort. Finally, a comparative analysis of learning behavior patterns between students in cluster 1 vs. cluster 2 was performed. Eye-tracking technology makes it possible to record large data about the learning process. However, using it in natural educational settings currently requires teachers to have technological and data mining skills.