Characterization of physicochemical properties of puff pastry margarines and their relationship with final product quality was investigated. The experiment involved the addition of two margarine samples (MLT and MHT), with different trans and saturated fatty acids content, in different quantities (35%, 45%, and 55% on flour basis) and with different relaxation time between laminating (15, 30, and 45 min). The results showed that MLT margarine (low trans) had statistically significantly lower solid fat content, firmness, and approximately 1.5 times lower shear stress compared with MHT margarine. Inadequate physical characteristics of MLT margarine, regardless of the independent variables, lead to bad quality of obtained puff pastry, while MHT margarine, despite the high content of TFA, enables obtaining pastry with good sensory quality. The obtained results indicate that the quantity of puff pastry margarine and the relaxation time need to be adjusted to the physicochemical characteristics of the margarine. Practical applications Bakery products such as puff pastry are products with high energy value as a consequence of high fat content that has a crucial role in the formation of product flaky texture. The puff pastry margarines are commonly obtained by partial hydrogenation and thus, these fats contain significant amounts of trans and saturated fatty acids that are harmful to health. Considering previously stated facts, the incorporation of low trans margarine in puff pastry is a new challenge in developing a novel healthier product. This study explores the application of low and high trans margarine, at different substitution levels and different relaxation time, as well as the correlation between margarine physicochemical properties and quality properties of puff pastry.