Abstract: Hybrid metric-Palatini gravity is a recently proposed theory, consisting of the superposition of the metric Einstein-Hilbert Lagrangian with an f(R) term constructed a la Palatini. The theory predicts the existence of a long-range scalar field, which passes the Solar System observational constraints, even if the scalar field is very light, and modifies the cosmological and galactic dynamics. Thus, the theory opens new possibilities to approach, in the same theoretical framework, the problems of both dark energy and dark matter. In this work, we consider the generalized virial theorem in the scalar-tensor representation of the hybrid metric-Palatini gravity. More specifically, taking into account the relativistic collisionless Boltzmann equation, we show that the supplementary geometric terms in the gravitational field equations provide an effective contribution to the gravitational potential energy. We show that the total virial mass is proportional to the effective mass associated with the new terms generated by the effective scalar field, and the baryonic mass. In addition to this, we also consider astrophysical applications of the model and show that the model predicts that the mass associated to the scalar field and its effects extend beyond the virial radius of the clusters of galaxies. In the context of the galaxy cluster velocity dispersion profiles predicted by the hybrid metric-Palatini model, the generalized virial theorem can be an efficient tool in observationally testing the viability of this class of generalized gravity models.