This study examines changes in gill Na+,K+-ATPase (NKA) α- and β-subunit isoforms, Na+,K+,2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR I and II) in anadromous and landlocked strains of Atlantic salmon during parr-smolt transformation, and after seawater (SW) transfer in May/June. Gill NKA activity increased from February through April, May and June among both strains in freshwater (FW), with peak enzyme activity in the landlocked salmon being 50% below that of the anadromous fish in May and June. Gill NKA-α1b, -α3, -β₁ and NKCC mRNA levels in anadromous salmon increased transiently, reaching peak levels in smolts in April/May, whereas no similar smolt-related upregulation of these transcripts occurred in juvenile landlocked salmon. Gill NKA-α1a mRNA decreased significantly in anadromous salmon from February through June, whereas α1a levels in landlocked salmon, after an initial decrease in April, remained significantly higher than those of the anadromous smolts in May and June. Following SW transfer, gill NKA-α1b and NKCC mRNA increased in both strains, whereas NKA-α1a decreased. Both strains exhibited a transient increase in gill NKA α-protein abundance, with peak levels in May. Gill α-protein abundance was lower in SW than corresponding FW values in June. Gill NKCC protein abundance increased transiently in anadromous fish, with peak levels in May, whereas a slight increase was observed in landlocked salmon in May, increasing to peak levels in June. Gill CFTR I mRNA levels increased significantly from February to April in both strains, followed by a slight, though not significant increase in May and June. CFTR I mRNA levels were significantly lower in landlocked than anadromous salmon in April/June. Gill CFTR II mRNA levels did not change significantly in either strain. Our findings demonstrates that differential expression of gill NKA-α1a, -α1b and -α3 isoforms may be important for potential functional differences in NKA, both during preparatory development and during salinity adjustments in salmon. Furthermore, landlocked salmon have lost some of the unique preparatory upregulation of gill NKA, NKCC and, to some extent, CFTR anion channel associated with the development of hypo-osmoregulatory ability in anadromous salmon.