Objective: This study aimed to investigate and compare the sperm acquisition rates achieved by microdissection testicular sperm extraction in patients with different causes of non-obstructive azoospermia. Methods: The medical records of 225 non-obstructive azoospermia patients who underwent microdissection testicular sperm extraction after January 2018 in the Reproductive Andrology Department of the Reproductive Hospital affiliated to Jiangxi University of Traditional Chinese Medicine were retrospectively analyzed. We investigated whether age, testicular volume, reproductive endocrine hormone levels, and etiological differences were correlated with sperm acquisition rates. Results: The patients were divided into two groups: those from whom sperm was successfully acquired and those from whom it was not. In the sperm acquisition group, there were 107 patients; in the non-acquisition group, there were 118 patients. There were no significant differences in age, testicular volume, endocrine hormone levels, body mass index, or inhibin B antigen levels between the two groups. A significant difference was found between the acquisition group (2.32±1.334) and the non-acquisition group (3.16±1.680) in levels of anti-Müllerian hormone (p<0.01). The patients were then classified based on the etiology of their non-obstructive azoospermia. Those patients whose condition was caused by a history of mumps, surgery for cryptorchidism, Klinefelter syndrome, and AZFc deletion had significantly higher sperm retrieval rates than patients with idiopathic non-obstructive azoospermia. Conclusion: Microdissection testicular sperm extraction is an established and effective treatment for infertility resulting from non-obstructive azoospermia. No correlation was found between testicular sperm extraction rates and age, obesity, testicular volume, endocrine hormone levels, or inhibin B levels. Anti-Müllerian hormone can be used to predict sperm retrieval rates but should be used alongside other indicators where possible. Sperm acquisition rates were found to be significantly correlated to the etiological origins of non-obstructive azoospermia. This can be used to predict the surgical sperm acquisition rates of non-obstructive azoospermia patients.