This study examined morphological, physiological and molecular indicators of smoltification in Atlantic salmon (Salmo salar) juveniles in a flow-through (FT) and recirculating aquaculture system (RAS). Fish were exposed to 24-h light to initiate smoltification, for 5 (FT) and 7 (RAS) weeks prior to transfer from freshwater (FW) to seawater (SW) and were sampled weekly preceding and following SW transfer. Mass, length, condition factor, plasma chloride, gill Na+/K+-ATPase (NKA) activity and expression of salinity-specific isoforms of NKA mRNA transcripts were monitored. Fish raised in FT had significantly lower specific growth rate (SGR) in FW than in SW and showed a significant 5-6-fold increase in gill NKA activity, and high plasma chloride levels after transfer to SW. These fish also exhibited no significant reduction in relative mRNA expression of NKAα-1a in FW but a sharp significant downregulation post-SW transfer. No significant increase in NKAα-1b was seen until week 8 (3 weeks post-SW transfer). The log2 ratio of NKAα-1b to NKAα-1a showed a significant 8-fold increase throughout the experiment. Fish raised in the RAS had significantly higher SGR in FW than SW, and showed significantly higher plasma chloride in saltwater challenged fish compared to the freshwater control at all weeks during the FW phase. Fish had a 50% higher initial NKA activity than in FT, increasing significantly 2-3-fold and showed an immediate down-regulation of NKAα-1a after exposure to 24-hr light in FW, and a further reduction after SW-transfer. There was no significant increase in NKAα-1b in the RAS-raised fish for the duration of the experiment, and there was a significant 8-fold increase in log2 ratio of NKAα-1b to NKAα-1a. Whilst there were too many varying factors to statistically compare hatchery type in this study, it’s evident that there are potentially system-related effects worthy of future investigation.