This study investigated the variation in electronic portal imager pixel scale at the isocenter plane for Elekta Agility linear accelerators. An in-house MATLAB script was written to process and calculate the pixel scale based on a metal calibration plate supplied by Elekta. Eight pixel plates were compared and found to have manufacturing tolerances within 0.1 mm of nominal dimensions. The impact of these variations on pixel scale factor was negligible, and plates could be used interchangeably. Uncertainties from other parameters such as source-to-surface distance and user variability summed to a combined uncertainty of 0.0003 mm/pixel, compared to a pixel scale range of 0.003 mm/pixel measured across 10machines. Most of the inter-machine variation was shown to be attributable to differences in source-to-panel distance. Other factors such as focal spot size and shape, electronic portal imager manufacturing consistency, panel sag, and setup errors may account for the residual variation. Individual characterization of machine and imaging panel pixel scale factors is important to ensure accurate geometric information is derived from electronic portal images, which is critical where the portal imager is used for multi-leaf collimator calibration or other clinical tasks.