Adeno-associated virus (AAV) are potent vectors to achieve treatment against hearing loss resulting from genetic defects. However, the effects of delivery routes and the corresponding transduction efficiencies for clinical applications remain elusive. In this study, we screened AAV vectors of three serotypes (AAV 8 and 9 and Anc80L65) into the inner ears of adult normal guinea pigs through trans-stapes (oval window) and trans-round window delivery routes in vivo. Trans-stapes route is akin to stape surgeries in humans. Then, auditory brainstem response (ABR) measurements were conducted to evaluate postoperative hearing, and inner ear tissues were harvested for transduction efficiency analysis. Results showed that AAV8 targeted partial inner hair cells (IHCs) in cochlear basal turn; AAV9 targeted IHCs in cochlear basal and second turn, also a part of vestibular hair cells (VHCs). In contrast, Anc80L65 contributed to green fluorescent proteins (GFP) signals of 80 - 95% IHCs and 67 - 91% outer hair cells (OHCs), as well as 69% VHCs through the trans-round window route, with 15-20 decibel (dB) ABR threshold shifts. And, through the trans-stapes (oval window) route, there were 71 - 90% IHCs and 42 - 81% OHCs, along with 64% VHCs demonstrating GFP positive, and the ABR threshold shifts were within 10 dB. This study revealed AAV could be efficiently delivered into mammalian inner ear cells in vivo through the trans-stapes (oval window) route with postoperative hearing preservation, and both delivery routes showed promise of virus-based clinical translation of hearing impairment treatment.