This paper introduced 7 cases of aquatic animals with relatively high difficulty using short-term injection method for chromosome rapid preparation. Methods: 7 cases of aquatic animals with great difficulty were used as materials to prepare chromosomes by in vivo injection of PHA and colchicine and air drying. The results showed that the optimal injection sites were different for different aquatic animals with different the injection sites, concentrations and the interval time. This paper listed the best conditions suitable for each aquatic animal, and established a rapid method for the preparation of aquatic animals' chromosomes in vitro. Conclusion: This method has some advantages, such as tissue-specificity, no need for aseptic operation, simple equipment, simple operation, rapid (2-4h), easy to manage, good stability, repeatability and good effect, which can be used for the rapid preparation of all aquatic animal chromosomes. The prepared chromosomes can be used for karyotype analysis, chromosome banding treatment, sex identification and FISH analysis. [Chinese Fishery Quality and Standards,2023,13(1):19-25]