Ponto-Caspian gobies have invaded many river systems in the northern hemisphere, but their colonization patterns often remain unrecognized at the early stages of invasion. Here, we analyzed the ongoing invasion of the round goby Neogobius melanostomus (Pallas, 1814) in the Elbe River (Germany) by combining literature research with electrofishing and environmental DNA (eDNA) sampling. Along the surveyed 517 km, abundances peaked in the upper and lower sections, while low or no goby abundances were detected in the middle section. Combining the results of the field survey and literature data, we inferred four dispersal modes, jointly appearing during the colonization. (1) Human mediated transport, which was responsible for the initial colonization of the tidal Elbe and for a jumpy expansion to the upstream sections. (2) Fast downstream colonization, which was particularly observed for juvenile individuals. (3) Slow upstream migration, which was also recorded for another goby species, the bighead goby Ponticola kessleri (Günther, 1861) and (4) Lateral invasion via a major shipping canal in the middle river section. The eDNA analyses generally supported these conclusions and showed a high sensitivity and thus increased detection strength at low goby densities. Our study showed that the location of the first occurrence, as well as the dispersal mode, strongly determines the invasion process of non-native gobies. © 2020, Springer Nature Switzerland AG.