The scattering mechanisms governing the transport properties of high mobility AlInN/AlN/GaN two-dimensional electron gas (2DEG) heterostructures with various AIN spacer layer thicknesses from zero to 2 nm were presented. The major scattering processes including acoustic and optical phonons, ionized impurity, interface roughness, dislocation and alloy disorder were applied to the temperature-dependent mobility data. It was found that scattering due mainly to alloy disorder limits the electron mobility for samples having spacer layer thicknesses up to 0.3 nm. On the other hand, alloy scattering is greatly reduced as the AlN spacer layer thickness increases further, and hence the combination of acoustic, optical and interface roughness become operative with different degrees of effectiveness over different temperature ranges. The room-temperature electron mobility was observed to increase gradually as the AlN spacer layer increases. A peak electron mobility of 1630 cm2 V−1 s−1 was realized for the sample consisting of a 1 nm AlN spacer layer. Then, the electron mobility decreased for the sample with 2 nm AlN. Moreover, the measured 2DEG densities were also compared with the theoretical predictions, which include both piezoelectric and spontaneous polarization components existing at AlN/GaN interfaces. The experimental sheet carrier densities for all AlInN/AlN/GaN HEMT structures were found to be in excellent agreement with the theoretical predictions when the parasitic (unintentional) GaN layer deposited between AlN and AlInN was taken into account. From these analyses, 1 nm AlN spacer layer thickness is found to be the optimum thickness required for high electron mobility and hence low sheet resistance once the sheet carrier density is increased to the theoretically expected value for the sample without unintentional GaN layer.