Smoltification of anadromous salmonids is a complex physiological process required for migration from freshwater to seawater environments. This process includes changes in fish condition factor, body color, behavior, and molecular profiles of muscle, brain, gill, and plasma, among other tissues. For wild fish, the transition to higher salinity environments is often gradual, with various amounts of time spent in estuaries prior to full seawater migration. However, smolts produced for aquaculture in land-based, freshwater hatcheries are transferred directly to seawater without brackish acclimation. This transfer also includes additional stressors such as crowding, transport, and handling. Thus, seawater transfer of salmonids in aquaculture is a key event that, if done sub-optimally, can lead to compromised immunity, lower osmoregulatory capacity, stunted growth, and increased rates of mortality. Several biochemical tests based on Na+/K+ ATPase (NKA) activity and mRNA abundance of NKA isoforms and related cotransporters are used by the industry to predict the readiness of smolts for seawater introduction. In the present study, a novel mRNA target for predicting Atlantic salmon smolt readiness is reported and benchmarked against industry standard markers. Multiple studies examining the expression of these genes from non-lethally sampled gill lamellae using RT-qPCR show that the predicative power in determining the life stage, smolt readiness, and response to environmental factors influencing smoltification (e.g. photomanipulation, functional feeds, etc.) are improved using the novel smolt gene. Furthermore, introduction of smolts to different salinities resulted in dose-response expression curves of the novel gene, with 100- and 300-fold increases in expression three days and three weeks post-seawater (25 ppt) transfer. This work demonstrates improvements to traditional smolt test markers and suggests that the use of several genes may offer improved insights on Atlantic salmon transfer to seawater.