ABSTRACT.Purpose: To test the validity of the EYESI SURGICAL SIMULATOR as an assessment tool in a virtual reality vitreoretinal training programme. Methods: In collaboration with an experienced vitreoretinal surgeon, a virtual vitreoretinal training programme was composed on the EYESI SURGICAL SIMULA-TOR, software version 2.9.2 (VRmagic GmbH, Manheim, Germany). It was completed twice by three groups: 20 medical students, ten residents of ophthalmology and five trained vitreoretinal surgeons. The programme contained six training modules: navigation level 2 (Nav2), forceps training level 5 (ForT5), bimanual training level 3 (BimT3), laser coagulation level 3 (LasC3), posterior hyaloid level 3 (PostH3) and internal limiting membrane peeling level 3 (ILMP3). The scores in each module were assessed from two to five different factors (tissue treatment, efficiency, target achievement, instrument handling and microscope handling), and it was possible to achieve 100 points in each module. Results: At the final training session, the highest overall median score was found for the vitreoretinal surgeons (vitreoretinal surgeons: 434 points, residents: 394.5 points, medical students: 272.5 points, p < 0.01). This was also found in four of the six modules. These were Nav2 (p = 0.03), BimT3 (p < 0.01), PostH3 (p < 0.01) and ILMP3 (p < 0.01). On the other hand, the three groups did not differ regarding ForT5 (p = 0.16) or LasC3 (p = 0.75). Conclusions: We developed a training programme with validity for the EYESI SURGICAL SIMULATOR as an assessment tool for overall score and for four of six vitreoretinal modules. These findings could potentially make the programme a useful tool in the training of future vitreoretinal surgeons.