The movements of organisms and the resultant flows of ecosystem services are strongly shaped by landscape connectivity. Studies of urban ecosystems have relied on two-dimensional (2D) measures of greenspace structure to calculate connectivity. It is now possible to explore three-dimensional (3D) connectivity in urban vegetation using waveform lidar technology that measures the full 3D structure of the canopy. Making use of this technology, here we evaluate urban greenspace 3D connectivity, taking into account the full vertical stratification of the vegetation. Using three towns in southern England, UK, all with varying greenspace structures, we describe and compare the structural and functional connectivity using both traditional 2D greenspace models and waveform lidar-generated vegetation strata (namely, grass, shrubs and trees). Measures of connectivity derived from 3D greenspace are lower than those derived from 2D models, as the latter assumes that all vertical vegetation strata are connected, which is rarely true. Fragmented landscapes that have more complex 3D vegetation showed greater functional connectivity and we found highest 2D to 3D functional connectivity biases for short dispersal capacities of organisms (6 m to 16 m). These findings are particularly pertinent in urban systems where the distribution of greenspace is critical for delivery of ecosystem services.