This Paper presents a comparison among four brain regions on the basis of cognitive load (Mental effort) imposed by the working memory during 2D multimedia animations learning. At present, 2D multimedia animations have become the popular learning style in educational field. This study will investigate which brain area require more cognitive load during multimedia animations learning. Electroencephalography (EEG) is one of the physiological methods that are used to measure the brain activity during any cognitive task. We have used EEG to investigate the cognitive activity of 2D multimedia animations during learning. One of the EEG waves, Alpha's Power recorded sensitive during alert state while facing task difficulty. These are the prominent brain waves to measure the cognitive load during any cognitive task. In this study, behavior of Alpha waves has been analyzed in four different brain regions to find the more cognitive load regions. For that analysis, five healthy male adults have been recruited and a 2D multimedia animation on biology has been shown to them. Results showed a decrease in power spectral density (PSD) of Alpha waves for all brain regions. Different responses are recorded across these four brain regions. Parietal and occipital regions are associated with visual information processing and interpretation so a high suppression of Alpha band power has been noticed during the 2D multimedia aid. © 2015 IEEE.