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In the 250 Wt RTPV (radioisotope thermophoto-voltaic) system, the heat source operates at a temperature above 1 000 ℃, which requires a long term stable thermal insulation devices. In this paper, three types of non-sealed high temperature MLI (multilayer insulator) were prepared by using spike, stainless steel wire mesh and aerogel as separator respectively. Each type was installed and tested in the RTPV prototype separately. The three MLIs are in the same size of 187 mm long, 183 mm wide and 50 mm thick. In MLI with spike separator, 50 metal foil reflectors were pierced several small holes, the spikes from the holes were used as the separator to make the adjacent reflectors in point contact. In MLI with stainless steel wire mesh separator, 50 stainless steel wire meshes were spot weld on each of the metal foil reflectors as the separator to make the adjacent reflectors in line contact. In MLI with aerogel separator, 18 polished metal plates were used as the reflectors, and slender strip aerogels in size of 50 mm×10 mm×2 mm were installed on four corners between adjacent reflectors as the separator. The main configuration of RTPV prototype tested in this paper was as follow. 1) An electric heating source with the size of 99 mm×95 mm×55 mm was used to simulate the radioisotope heat source. The heat source was supported in RTPV center by seven ceramic nails and coated with tungsten alloy radiation emitter. 2) Four optical filters were placed 34 mm away from the heat source and supported by four inner brackets. 3) Photovoltaic cells were installed on the side walls inside the housing with a distance of 14 mm from the optical filter. 4) Two high temperature MLIs were installed on the upper and lower end covers inside the housing. 5) Cold plates were used on the side walls outside the housing. Vacuum thermal performances of RTPV with different MLIs were carried out on a test platform. Based on the test results, some conclusions come out as follows. 1) The aerogel separator has the best performance in controllability of the assembly separating effect, which leads to the best thermal insulation performance consistency between different batches. 2) When the assembly separating effect is well controlled, the high temperature MLI with spike separator has a better insulation performance than stainless steel wire mesh separator. 3) The optical cavity inside the RTPV prototype is consisted of high-temperature MLI and the optical filters. A better closure of the optical cavity significantly contributes to the improvement of the heat utilization, and accesses to a higher heat source temperature. The optical cavity has the best closure performance when using the high temperature MLI with aerogel separator. While the RTPV prototype with aerogel separator input power is 250 Wt, the heat source temperature reaches 1 038 ℃.