ARTICLE Excited-state spin-resonance spectroscopy of V  B defect centers in hexagonal boron nitride Nikhil Mathur 1,10 , Arunabh Mukherjee 2,10 , Xingyu Gao 3 , Jialun Luo 4 , Brendan A. McCullian 1 , Tongcang Li 3,5 , A. Nick Vamivakas 2,6,7,8,11 ✉ & Gregory D. Fuchs 1,9,11 ✉ The recently discovered spin-active boron vacancy (V  B ) defect center in hexagonal boron nitride (hBN) has high contrast optically-detected magnetic resonance (ODMR) at room- temperature, with a spin-triplet ground-state that shows promise as a quantum sensor. Here we report temperature-dependent ODMR spectroscopy to probe spin within the orbital excited-state. Our experiments determine the excited-state spin Hamiltonian, including a room-temperature zero-field splitting of 2.1 GHz and a g-factor similar to that of the ground- state. We confirm that the resonance is associated with spin rotation in the excited-state using pulsed ODMR measurements, and we observe Zeeman-mediated level anti-crossings in both the orbital ground- and excited-state. Our observation of a single set of excited-state spin-triplet resonance from 10 to 300 K is suggestive of symmetry-lowering of the defect system from D 3h to C 2v . Additionally, the excited-state ODMR has strong temperature dependence of both contrast and transverse anisotropy splitting, enabling promising avenues for quantum sensing. https://doi.org/10.1038/s41467-022-30772-z OPEN 1 School of Applied and Engineering Physics, Cornell University, Ithaca, NY, USA. 2 The Institute of Optics, University of Rochester, Rochester, NY, USA. 3 Department of Physics and Astronomy, Purdue University, West Lafayette, IN, USA. 4 Department of Physics, Cornell University, Ithaca, NY, USA. 5 Elmore Family School of Electrical and Computer Engineering, Purdue University, West Lafayette, IN, USA. 6 Materials Science, University of Rochester, Rochester, NY, USA. 7 Department of Physics and Astronomy, University of Rochester, Rochester, NY, USA. 8 Center for Coherence and Quantum Optics, University of Rochester, Rochester, NY, USA. 9 Kavli Institute at Cornell for Nanoscale Science, Ithaca, NY, USA. 10 These authors contributed equally: Nikhil Mathur, Arunabh Mukherjee. 11 These authors jointly supervised this work: A. Nick Vamivakas, Gregory D. Fuchs. ✉ email: nick.vamivakas@rochester.edu; gdf9@cornell.edu NATURE COMMUNICATIONS | (2022)13:3233 | https://doi.org/10.1038/s41467-022-30772-z | www.nature.com/naturecommunications 1 1234567890():,;