During the primary drying step of the freeze-drying process, mass transfer resistance strongly affects the product temperature, and consequently the final product quality. The main objective of this study was to evaluate the variability of the mass transfer resistance resulting from the dried product layer (R_p) in a manufacturing batch of vials, and its potential effect on the product temperature, from data obtained in a pilot scale freeze-dryer. Sublimation experiments were run at -25 °C and 10 Pa using two different freezing protocols: with spontaneous or controlled ice nucleation. Five repetitions of each condition were performed. Pressure rise test (PRT) and gravimetric methods were applied as complementary approaches to estimate R_p. The PRT allowed to assess variability of the evolution of R_p with the dried layer thickness between different experiments whereas the gravimetric method informed about R_p variability at a fixed time within the vial batch. A product temperature variability of approximately ± 4.4 °C was defined for a product dried layer thickness of 5 mm. The present approach can be used to estimate the risk of failure of the process due to mass transfer variability when designing freeze-drying cycle.