Objective?To systematically evaluate the neuronal transduction efficiency of AAV capsid variants including AAV-PHP.B and AAV-PHP.eB in the brain of cynomolgus monkeys (Macaca fascicularis), and to analyze the effects of different injection doses and routes on whole-brain infection.?Methods?Recombinant AAVs (rAAVs) carrying the hSyn-EYFP expression cassette packaged into AAV9, AAV-PHP.B, and AAV-PHP.eB capsids were administered to cynomolgus monkeys via different routes (intracerebroventricular, ICV; intracisternal magna, ICM; intravenous, IV) and at different doses. After 6 weeks of transgene expression, the animals were perfused, and brain tissues were collected for cryosectioning and immunofluorescence staining. The ratio of EYFP and NeuN co-labeled cells to the total NeuN-positive cells in regions including the prefrontal cortex, caudate/putamen nucleus, hippocampus, and thalamus was quantified to assess neuronal transduction efficiency.?Results?Following ICV injection, AAV-PHP.B at a low dose (1×1013 vg·kg-1) demonstrated significantly higher neuronal transduction efficiency than AAV9 in the prefrontal cortex (8.4% vs 1.2%) and caudate/putamen nucleus (6.3% vs 0.5%). Increasing the AAV-PHP.B dose to 5×1013 vg·kg-1 further enhanced its efficiency in the caudate/putamen nucleus. Combined ICV and ICM injection synergistically enhanced the whole-brain transduction of AAV-PHP.B, particularly in the prefrontal cortex (14.7%) and thalamus (2.6%). When delivered via the combined route, AAV-PHP.eB showed significantly superior transduction efficiency in deep brain structures like the hippocampus and thalamus compared to AAV-PHP.B. In contrast, intravenous injection of AAV-PHP.eB failed to achieve efficient transduction (<1%) in all brain regions examined.?Conclusions?The AAV-PHP.B and AAV-PHP.eB capsids exhibit superior transduction capabilities in the cynomolgus monkey brain compared to AAV9. The combined ICV and ICM injection strategy represents a highly effective approach for achieving widespread neuronal transduction throughout the brain. This study provides crucial experimental evidence for selecting appropriate AAV delivery tools and optimizing administration protocols for non-human primate studies and future clinical translation.