Abstract This article describes a novel method for quantifying fixation disparity and evaluates its role in visuospatial cognition during an authentic learning task, specifically, the determination of molecule chirality in organic chemistry involving mental rotation and pattern comparison. The first study examined the influence of molecular model dimensionality (2D vs. 3D) on chirality determination performance and visual attention of 55 participants. The second study explored how the sustained playing of the tile-matching game Mahjong, a pattern comparison game, can affect visual attention and visuospatial performance during the chirality determination task of 59 participants. Fixation disparity was one of the eye tracking variables explored. Both studies revealed that (1) individuals with higher fixation disparity underperformed on the chirality task, which involves mental rotation and pattern comparison, and (2) fixation disparity improved over time in participants who played Mahjong. This work provides important implications for using fixation disparity as a possible biomarker of visuospatial performance.