With the increasing availability of fast-growing Eucalyptus plantation logs in Australia in recent years, the timber manufacturing sector has become interested in discovering the opportunities of producing value-added timber products from this resource. Cross-laminated timber (CLT) could be a potential sustainable product recovered from this resource and supply material for commercial buildings. Shear of the inner cross-laminates, known as rolling shear, is one of the governing factors in serviceability and limit state design for this product under out-of-plane loading. This study evaluated the rolling shear (RS) properties of CLT with heterogonous layup configurations using different structural grade Eucalyptus nitens (E. nitens) timber under the planar shear test. Based on the results, Gr and tr values were shown to be significantly correlated with the density of the CLT panel. There was also a positive correlation between the RS modulus and MOR of the CLT panel. The specimens with high MOE in the top and bottom layers indicated the highest tr and Fmax values. This indicated that using high-grade boards in the top and bottom lamellae plays an important role in increasing the RS strength, whereas using them in the cross-layer has a positive contribution in increasing shear modulus. The maximum observed RS strength and modulus ranged from 2.8–3.4 MPa and 54.3–67.9 MPa, respectively, exceeding the RS characteristic values of the resource. The results obtained in this study were comparable to those recommended in European standards for softwood CLT, demonstrating the potential use for eucalypt timber boards in CLT production. This paper provides an important insight into supporting the potential engineering applications of CLT panel products fabricated with eucalypt plantation.