Abstract: Quercus aquifolioides as a dominant broad-leaved tree species in southeastern Tibet, plays a crucial role in soil and water conservation, as well as water source retention in high-altitude regions. Currently, seed insect infestation is a major constraint affecting natural regeneration in secondary forest, seedling cultivation, and industrial utilization. This study investigated the seed insect infestation status of Q. aquifolioides and its impact on seed quality across different altitudes (3000–3500 m) on Sejila Mountain. The aim was to clarify the extent and characteristics of pest damage to provide a foundation for the conservation and utilization research of Q. aquifolioides resources. The results demonstrated that: (1) The infestation rate decreased significantly with increasing altitude. In the same period, the infestation rate was usually the highest at 3000 m (reaching 95.05% in mid-August) and the lowest at 3500 m (80.08% in mid-August). Concurrently, the infestation rate increased with delayed collection date. At the same altitude, the infestation rate increased progressively from mid-August to mid-September (increasing from 95.05% to 98.90%). (2) The seed quality exhibited a U-shaped pattern across the altitude gradient, being lowest at 3200 m and highest at 3500 m. (3) The thousand-seed weight and seed volume fluctuated with collection time and altitude. In mid-August, the thousand-seed weight increased with altitude (850.78 g at 3000 m vs. 1152.08 g at 3500 m). However, in mid-September, the thousand-seed weight at the mid-altitude (3200 m) decreased drastically (640.63 g). Meanwhile, the seed volume peaked at 3400 m in mid-August (2.628 cm³), and decreased significantly at mid-to-high altitudes (3200–3300 m) in mid-September. (4) The seedling emergence rate increased with altitude, with the highest at 3500 m (reaching 35% in mid-August), and the emergence rate was significantly lower at low (3000–3100 m) and middle (3200 m) altitude. Seeds collected in mid-September generally exhibited decreased emergence rate.