Background: Non-obstructive azoospermia (NOA), marked by impaired spermatogenesis, poses challenges in assisted reproduction. Limited data exist comparing chromosomal integrity of testicular versus ejaculated sperm. This study aimed to compare embryo morphokinetics, fertilization abnormalities, and PGT-A outcomes between embryos from ejaculated and testicular sperm in NOA cases. Subjects and Methods: This retrospective study analyzed 397 patients from two IVF centers (2015–2023), with 317 using ejaculated sperm and 80 using testicular sperm from NOA patients. Fertilization patterns (2PN, 1PN, ≥3PN), embryo morphokinetics, and aneuploidy rates were assessed. Logistic regression examined factors influencing aneuploidy including male and female age, recurrent implantation failure (RIF), hormone levels, and oocyte quality, while chi-square and t-tests compared groups, with significance at p<0.05. Results: Embryos derived from testicular sperm developed faster than those from ejaculated sperm (p<0.05). The 2PN fertilization rate was significantly lower, while the 1PN rate was higher in the testicular sperm group (10.1% vs. 16.4%, p=0.020). The rates of ≥3PN anomalies and embryo aneuploidy were similar between groups (p>0.05). Logistic regression identified male age (p=0.001), female age (p=0.007), and RIF (p=0.047) as significant predictors of aneuploidy. Conclusion: Our study identified advanced parental age and RIF as key predictors of embryo aneuploidy. PGT-A may improve outcomes, especially for older patients or those with RIF, regardless of sperm origin. Embryos from testicular sperm in NOA patients develop faster morphokinetically but show a higher rate of 1PN fertilization than those from ejaculated sperm.