During the lyophilization process, formulations containing protein, bulking agent, or lyoprotectant form a dry product layer that can affect the transport of sublimed water vapor. We carried out an investigation of the primary drying segment of lyophilization to evaluate the relationship between the resistance to water vapor flow through the dried layer and the microstructure of the dried cake. Recombinant humanized antibody HER2 (rhuMAb HER2) formulated in trehalose was studied, as were protein-free formulations containing trehalose and sucrose. Sublimation rate and product temperature data were used to compute the resistance to mass transfer. Dried cake structure was examined by scanning electron microscopy and a novel fluorescence microscopy method. Collapse temperatures were determined by freeze-drying microscopy. Mass transfer resistance was found to decrease with increases in temperature for each material. Resistance also depended on composition, decreasing in the formulation series, rhuMAb HER2, trehalose, sucrose. The lyophilized material consisted of porous cakes, with a distinct denser region at the top. Formulation and temperature affected the microstructure of the dried cakes. The formulated trehalose and sucrose were seen, by both microscopy techniques, to possess small (2–20 µm) holes in their platelike structures after lyophilization. The quantity of holes was higher for material dried at higher temperature. The collapse temperature (Tc) of a material appeared to play a role in the process, as lower Tc was correlated with lower resistance and a greater extent of holes. Our results are consistent with the theory that lower resistance to water vapor flow in the primary drying stage of lyophilization may be due to small-scale product collapse.