Establishing a feasible disinfection system for obtaining the germfree mature lotus (Nelumbo) embryos as explants and understanding the differences of in vitro growth of lotus from different provenances is the basis for material selection and evaluation method to construct an efficient in vitro culture and genetic transformation system. Plant preservative mixture (PPM) at different concentrations was used in the pre-culture stage to develop a suitable disinfection system of lotus seeds and a method of acquiring the germ-free lotus embryos. Through this sterilization approach, the in vitro cultured seedlings of lotus from 9 worldwide provenances were successfully obtained. Based on the morphological indexes at different culture periods, the membership function method was used to evaluate the growth differences under the in vitro culture conditions. The results showed that the ideal disinfection system for lotus seeds was 75% alcohol 30 s + 2% PPM 2 h + 0.05% PPM 3-5 d soaking during germination. The in vitro growth differences of the seedlings from the tested lotuses of 8 provenances were mainly observed in the later culture stage, such as the number of leaves, the length and thickness of rhizomes, the number of roots and lateral shoots. The seedlings of lotus from Vietnam and Thailand had the highest growth potential and higher reproduction rate under the same culture conditions. The pre-culture method by soaking lotus seeds with PPM to successfully obtain germ-free lotus embryos has solved the technique problem in disinfecting lotus seeds in the past. The in vitro clones of lotus embryos from the tropical or subtropical regions in both Vietnam and Thailand have stronger growth capacity and therefore are the materials more ideal for establishing regeneration and genetic transformation system of Nelumbo.