Conventional ophthalmic eye drops are limited by their rapid elimination rate and short time of action. Ion exchange resin has been used to achieve sustained ocular drug delivery but the high selectivity of drug molecules restricts its broad application. In situ gel system seems to be a good strategy to address these problems but the influence of in situ gel type on the sustained release behavior and tissue distribution after ocular application is unclear. Therefore, in this study, using betaxolol hydrochloride as a model drug, poloxamer 407 and methylcellulose as the carriers, two thermosensitive in situ gel systems were prepared and characterized. Influence of formulation composition type and concentration on in vitro drug release was studied. Tissue distribution after ocular delivery of two different thermosensitive in situ gels was studied and compared with commercial BH eye drop (Betoptic S®). In vitro studies demonstrated that addition of 4% HPMC 606W in 15% P407 solution and 5% PEG4000 in 2% MC solution obtained gels with appropriate gelation temperature and similar sustained drug release rate. In vivo tissue distribution study indicated that they presented similar drug concentration in cornea, iris-ciliary and aqueous humor irrespective of gel type, with higher drug concentration achieved after 4 h compared to the commercial resin suspension eye drops. The AUC and MRT of the two in situ gel eye drops were 2 times higher than that of the commercial resin suspension eye drops in cornea. In conclusion, the two thermosensitive in situ gels have prolonged drug release after ocular drug delivery compared with ion exchange resin eye drops, implying their potential applications in clinic with broad drug adoptability.