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Increasing colonization of non-native amphipod species in the River Thames, United Kingdom, has altered aquatic ecology and called existing management practices into question. We studied the distribution patterns of recent non-native (Dikerogammarus haemobaphes (Eichwald, 1841)), established non-native (Crangonyx pseudogracilis (Bousfield, 1958)), and native amphipod (Gammarus pulex (Linnaeus, 1758)) species, as well as habitat and human influences across 84 sites in the upper Thames catchment. Our findings showed widespread distribution and density of G. pulex relative to D. haemobaphes, suggesting that the full impact of the current spread has yet to be felt since its 2012 introduction. Different habitat utilization patterns are explained through habitat partitioning: both D. haemobaphes and C. pseudogracilis occupied vegetative habitats, not pebble/gravel habitats where the native G. pulex was most often found. The association between D. haemobaphes and boating presence implies that effective biosecurity would be best focused on boat traffic in the Thames and Cherwell rivers.