In euryhaline teleosts, Na+, K+-ATPase (NKA) and V-type H + -ATPase A (VHA A) are important ion-transporters located in cell membrane. Lipid rafts (LR) are plasma membrane microdomains enriched in cholesterol, sphingolipids, and proteins (e.g., flotillin). Flotillin is a LR-associated protein, commonly used as the LR marker. Previous mammalian studies showed that LR may play a crucial role in ion exchanges. Meanwhile, studies on mammals and rainbow trout showed that NKA were found to be present mainly in LR. However, little is known about LR in fish. Therefore, the present study aimed to investigate the involvement of branchial LR in osmoregulation of tilapia and milkfish, two euryhaline teleosts with different salinity preferences, by (i) extracting LR from the gills of euryhaline teleosts; (ii) detecting the abundance of LR marker protein (flotillin-2) and ion-transporters (NKA and VHA A) in branchial LR and non-LR of fresh water- and seawater-acclimated milkfish and tilapia. The results indicated that the protein abundance of LR marker, flotillin-2, changed with environmental salinities in branchial LR of tilapia. In addition, flotillin-2 and NKA were only found in LR in both tilapia and milkfish gills, while VHA A were mainly present in non-LR. Relative protein abundance of NKA was found to be significantly higher in gills of freshwater milkfish and seawater tilapia, while VHA A was significantly higher in gills of freshwater tilapia and milkfish. This study illustrated differential distribution and salinity-dependent expression of NKA and VHA A in cell membrane of gill tissues of euryhaline teleosts with different salinity preferences.