High-speed volumetric imaging of neural activity at cellular resolution is important to capture the emergent functional properties of neural circuits. While two-photon calcium imaging provides a power tool to study population activity in vivo, standard two-photon microscopes only image two-dimensional planes. Expanding it to three-dimensions while maintaining a high spatiotemporal resolution appears necessary. Here, we extended the sequential volumetric two-photon imaging by a novel hybrid approach that acquires two volumes simultaneously through wavelength multiplexing, combining dual color excitation and emission with ETL/SLM-based fast z- scan approaches. We use 920 nm and 1064 nm lasers to image, at the same time, neurons labeled with GCaMP6 in mice cortical layer 2/3, and with jRGECO in layer 5, respectively. An electrically tunable lens or a spatial light modulator was implemented in the beam path to enable fast sequential or simultaneous imaging of different focal planes. Using this beam multiplexing strategy, we image the neuronal activity of cortical circuits at high speed in primary visual cortex from awake mice (600 μm deep volumes at 10 vol/s). We analyze the orientation tuning properties of cells in cortical columns, as well as the spatial structures of visually-evoked neuronal ensembles. We also simultaneously image functional correlations between presynaptic layer 1 axons and postsynaptic layer 2/3 neurons. Wavelength multiplexing enhances high-speed volumetric microscopy and can be combined with other multiplexing schemes to easily boost imaging throughput.1.