[Objectives] Sperm morphology is important in sperm competition and sperm evolution. The complex cloacal glands in both males and females of caudate amphibians with internal fertilization may be responsible for further packaging and modification of spermatogenesis, but little is known about the effects of these processes on sperm morphology and function. [Methods] In this study, the species Liangshantriton taliangensis which practices internal fertilization by transferring a spermatophore was studied. The morphological changes of the features of spermatozoa collected by 3 kinds of sampling methods (testes crashing, spermatophore, and female cloaca flushing) were systematically compared. Using t-test, t’-test, or Mann-Whitney U test, we analyzed the sperm morphological changes, and significant differences were set at P < 0.05. [Results] The sperm from the spermatophore and female cloaca flushing had a distinct neck, while the sperm from the testes had no such structure, but with cytoplasmic masses around the sperm head (Fig. 1). In addition, sperm shortens the acrosome and increases the proportion of sperm tail in the process of being passed out of the body by the testes (Mann-Whitney U test or t-test, P < 0.05) (Table 1), no significant change (Mann-Whitney U test or t-test, P > 0.05) in sperm morphology and measurement during the process from spermatophore to female body except for the further shortening of acrosome (t-test, P < 0.05) (Table 2). [Conclusion] These results suggest that sperm samples from the testis, spermatophore, or female ejaculatory cavity may show morphological and functional differences due to modification processes, which provide great options for the sampling method for the sperm biology or sperm conservation studies.