Two enantiomers (mirror images) can show drastically different behaviors, resulting in the enantiomers' identification and separation being in high demand in biomedical research and industry. Here, we introduce an optical approach in which, by using a tightly focused vector beam with radially varied polarizations, we realize the selective trapping of both enantiomeric forms. Numerical results show that such a focused field exhibits bifocal spot intensity distribution and can simultaneously stably trap one enantiomer in one focal spot and the other enantiomer in the other spot in three dimensions, achieving an effective separation of the chiral entities. The trapping distance and position of the enantiomeric pairs can be changed by separately varying the magnitude and sign of the polarization topological charge of the vector beam. And the difference in trapping potentials of the particles with different chirality provides a further identification of the chirality. Our theory indicates that the enantiomers' identification and separation can be mediated by the same incident beam, providing a possible route to detect, separate, and manipulate chiral objects at nanometer scales. (C) 2019 Optical Society of America