Increasing consumer interest in pulse flour-based food formulations has highlighted the importance of flour characterization based on the type (variety) of pulse and milling methods. The objective of the present study was to compare the microstructure of four different pulse flours (chickpea, navy bean, green lentil, and yellow pea) obtained from two milling methods (single-stage Ferkar mill and multi-stage roller mill). Therefore, in the present study, the particle size distribution, protein content, and microstructural characteristics of starch-protein matrices were investigated using laser diffraction, the Dumas method, and scanning electron microscopy. Micrographs depicted that protein bodies were finely distributed in Ferkar-milled flours and straight grade streams of roller-milled flours with similar protein content. The reduction flour stream (possessing the highest protein content) contained more damaged starches than the break flour stream (possessing the lowest protein content) when observed visually. Chickpea starch granules were observed to be highly porous as compared to the starch granules in other pulse flours. Traces of fiber particles were visible in green lentil flours obtained from roller and Ferkar mills. Conclusively, the microstructure and protein content of the pulse flours is influenced by the pulse-type and milling method. Enhanced ingredient functionality obtained from flour fractions will benefit food manufacturers to incorporate pulse flours in food formulations to enhance the nutritional and textural properties of end products.