This study aimed to develop reference materials (RMs) for mortar that can simulate the initial flow characteristics with constant quality over a long period. Through the previous research on the development of RMs for cement paste, the combination of limestone, glycerol, and water was used as the basic matrix for developing RMs for mortar in this study. In addition, glass beads of three particle sizes (0.5, 1.0, and 2.0 mm) and ISO standard sand were selected as tentative candidates to derive fine aggregate substitutes. The mixture of glass beads could simulate the initial flow characteristics of mortar, but under the same mixing ratio, replicates showed an unstable tendency to indicate inconsistent values due to the generation of electrostatic properties between materials and equipment. On the other hand, the mixture using ISO standard Sand not only simulates the constant flow characteristics for a long period of time, but also shows stable results with little error in replicates. Therefore, limestone, glycerol, ISO standard sand, and water were finally determined as components that met the required properties of RMs for mortar. The effect of each component on the flow characteristics of RMs was analyzed. It was found that glycerol increased the cohesion between the particles of standard sand, resulting in a constant increase both in the plastic viscosity and yield stress. Both limestone and standard sand had a dominant effect on the yield stress. The relationships between various mortar mixing ratios and the corresponding mixing ratios of RMs were established. In addition, the results of the verification experiment showed that the rheological properties of the RMs obtained through the relationships correlated with various water/cement ratios and the fine aggregate volume fractions of mortar obtained with same manner. In other words, the RMs for mortar developed in this study can be used as standard samples because they can simulate the initial flow characteristics of mortar of various mixing ratios for a long period without any chemical changes.