Species invasions are a global problem of increasing concern, especially in highly connected aquatic environments. Despite this, salinity conditions can pose physiological barriers to their spread and understanding them is important for management. In Scandinavia’s largest cargo port, the invasive round goby (Neogobius melanostomus), is established across a steep salinity gradient. We used 12 937 SNPs to identify the genetic origin and diversity of three sites along the salinity gradient and round goby from western, central and northern Baltic Sea, as well as north European rivers. Fish from two sites from the extreme ends of the gradient were also acclimated to freshwater and seawater, and tested for respiratory and osmoregulatory physiology. Fish from the high salinity environment in the outer port showed higher genetic diversity, and closer relatedness to the other regions, compared to fish from lower salinity upstream the river. Fish from the high salinity site also had higher maximum metabolic rate, fewer blood cells and lower blood Ca2+. Despite these genotypic and phenotypic differences, salinity acclimation affected fish from both sites in the same way: seawater increased the blood osmolality and Na+ levels, and freshwater increased the levels of the stress hormone cortisol. Our results show genotypic and phenotypic differences over short spatial scales across this steep salinity gradient. These patterns of the physiologically robust round goby are likely driven by multiple introductions into the high salinity site, and a process of sorting, likely based on behaviour or selection, along the gradient. Since this euryhaline fish risks spreading from this area, seascape genomics and phenotypic characterisation can inform management strategies even within an area as small as a coastal harbour inlet.