Elekta AQUA v2.02 software (Gantry Runout isocenter test) was investigated as a tool for verification of kilovoltage to megavoltage gantry radiation isocenter coincidence. AQUA reported megavoltage (6 MV) isocenter was independent of field size over the range 5 cm × 5 cm to 20 cm × 20 cm. For the 10 cm × 10 cm field size, standard deviation in AQUA reported 3D megavoltage (6MV) isocenter over ten consecutive deliveries was less than 0.04 mm for any axis. Compared to the full AQUA test delivery (Gantry Runout), the shorter AQUA test version (Gantry Runout short) gave a root mean square MV isocenter (± 1 SD) difference of 0.18 mm ± 0.08. Across 7 machines, root mean square differences between AQUA and PIPS PRO reported MV isocenter (for 6 MV and 6 MV FFF beams) was 0.1 mm ± 0.1 mm, with most of the difference observed in the gun-target (Y-axis). AQUA 6 MV isocentre position was offset to gantry relative to Elekta XVI customer acceptance test (CAT) workflow by 0.25 mm to 0.52 mm. For 6 MV FFF beams, AQUA reported an MV isocenter of between 0.27 and 0.39 mm offset to target relative to Elekta CAT.